IN SILICO STUDY OF EUCALYPTOL FROM EUCALYPTUS GLOBULUS LABILL. AGAINST ANGIOTENSIN-CONVERTING ENZYME AS AN ANTIHYPERTENSIVE IN COVID-19 COMORBID

Objective: This study aimed to determine the best compound from the 62 compounds of Eucalyptus globulus Labill. as an antihypertensive based on its interaction with angiotensin-converting enzyme (ACE) using the in silico study. Methods: The study was carried out in silico through molecular docking simulations, analysis of potential compounds using Lipinski’s rule, and ligand-based ADMET prediction on 62 compounds of the E. globulus . Results: It was found that eucalyptol (1,8-cineole) had the best interaction with the ACE as indicated by a bond energy value ( ∆G) of -6.40 kcal/mol with an inhibition constant of 20.82 µM, and interacted with key amino acid residues in captopril, namely HIS513, HIS353, TYR523, and ALA354. Eucalyptol also had good physicochemical properties by fulfilling Lipinski’s rule and had the best ADMET profile compared to other compounds. Conclusion: Eucalyptol was the best antihypertensive against ACE based on amino acid residue interaction, physicochemical properties, and ADMET profile.


INTRODUCTION
Coronavirus Disease 2019 (COVID- 19) is a viral infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and attacks the human respiratory system.The first case of COVID-19 was discovered in Wuhan, China at the end of the year 2019 and rapidly spread worldwide [1].The spread of COVID-19 cases to various countries, with a rapid increase in the number of events, led WHO to declare COVID-19 a pandemic on March 11, 2020.Data on confirmed cases of COVID-19 in the world continued to increase in 2021 and there is no sign of a downward trend in the number of cases [2].This pandemic decrease in 2022.
Based on the results of several studies that have been conducted show that factors such as age>65 y, being male, and having comorbid diseases are independent risk factors for increasing the severity of the disease and death from COVID-19.The results of clinical and epidemiological data analysis of COVID-19 show that 20-51% of COVID-19 patients have at least one comorbidity, such as hypertension (21.1%), cardiovascular disease (8.4%), diabetes (9.7%), and respiratory tract disease (1.5%) [3].Research by Ejaz H, et al. [4] found the mortality rate of patients with confirmed COVID-19 who had comorbidities in China, namely hypertension (9.5%), diabetes (7.4%), chronic obstructive pulmonary disease (COPD, 7.0%), cardiovascular disease (7.3%), liver disease (2.4%), obesity (13%), kidney disease (0.7%), and malignancy (2.0%).Other data from Italy found a mortality rate of COVID-19 infection with comorbid hypertension (73.8%), diabetes (35.5%),COPD (13.7%), cardiovascular disease (42.5%), liver disease (3.7%), obesity (8.5%), kidney disease (20.2%), and malignancy (5.0%).It can be concluded that one of the most common comorbid diseases suffered by confirmed COVID-19 patients is hypertension and also one of the comorbidities with the highest mortality rate.Among genetic reasons, the angiotensin II enzyme, which is produced as a result of the abnormal function of the reninangiotensin system, is reported as a major cause of hypertension.Angiotensin Converting Enzyme (ACE) is considered to play an important role in controlling hypertension.Therefore, ACE can be a potential therapeutic target in regulating the conversion of angiotensin I to angiotensin II and ultimately controlling hypertension [5].
Eucalyptol is an important component of the Eucalyptus globulus Labill and a study demonstrating that intravenous administration of eucalyptol significantly reduced blood pressure in awake and anesthetized rats.Measurements with isolated rat aortas showed that eucalyptol has a vasodilating effect, suggesting that the blood pressure-lowering effect may result from a decrease in peripheral vascular resistance due to the direct relaxation of vascular smooth muscle [6].Animal studies have shown that renin-angiotensin-aldosterone system (RAAS) inhibitors increase the expression of ACE2 in cardiac tissue [7], leading to concerns that hypertension may increase the interaction of the virus with host cells and worsen COVID-19.Hypertension is almost double the severity and mortality of COVID-19 [8,9].This study was conducted to determine the best compounds that have potential as antihypertensives from the 62 compounds in E. globulus.The activity was determined using in silico study based on the interaction of the 62 compounds with ACE and ADMET prediction.This study was important to do because hypertension increases the severity and mortality in COVID-19 patients.

Materials
The software in this study was hardware in the form of personal laptops with Intel(R) Core(TM) i5-8250U processor specifications @ 1.60GHz 1.80 GHz and RAM 4GB and software, such as BIOVIA Discovery Studio 2017® [10], AutoDock Tools® [11], ChemDraw, Chem 3D, and PreADMET 2.0 [12].The materials were ACE, which was downloaded via the Protein Data Bank (https://www.rcsb.org/)with the PDB code 2XY9 and the three-dimensional structure of 62 compounds of E. globulus prepared with the Chem 3D program.

Methods
This research was conducted in silico on the structure of the isolated compound from E. globulus against ACE (PDB ID: 2XY9) with the following stages, i. e, selection of test compounds using Lipinski's rule of five analysis, prediction of ADME, the toxicity of test compounds of E. globulus, and pharmacophore modeling.

RESULTS AND DISCUSSION
ACE with the PDB ID: 2XY9 was downloaded via the Protein Data Bank [13], and then prepared using the BIOVIA Discovery Studio

Fig. 1: Structure of ACE (A) and native ligand (B) which had been separated from ACE
Lipinski's rule is a rule for the physicochemical properties of a ligand so that the hydrophobic/hydrophilic character of a compound through the cell membrane for passive diffusion can be determined.Lipinski's rule can help in observing the permeability of a drug to the lipid bilayer of the target body.Lipinski's rule consists of four points, namely (1) molecular weight<500 Da; (2) Log P (partition coefficient)<5; (3) number of hydrogen bond donors<5; (4) the number of hydrogen bond acceptors is less than 10 [15].
If the test compound has a molecular weight>500 Da, it will be difficult for the compound to penetrate the cell membrane.A Log P value that is greater than 5 also indicates that the compound will be increasingly lipophilic which causes the compound to bind tightly to the membrane, making it difficult to recognize the target protein and is toxic.Donor hydrogen bonds in a compound will partition in solvents that have strong hydrogen bonds (such as water).Meanwhile, hydrogen bond acceptors affect permeability because they have more ability to interact well in solvents that have strong hydrogen bonds, such as water [16].About 62 compounds were docked and the results are presented in table 1 following Lipinski's Rule of Five.All the test compounds met the requirements and could be used as oral drug candidates and could be further investigated for their pharmacokinetic and toxicity profiles (table 1).In carrying out molecular docking, validation was required by redocking ACE with the native ligand that had been separated previously using the Autodock 4.2 program.The Root Mean Square Deviation (RMSD) value was used as a method validation parameter, where this value indicated a deviation from the measurement results when measurements were carried out repeatedly.The RMSD value of molecular docking indicated the deviation of the bond pose that occurs in the test ligand compared to the reference bond pose (download from PDB).The lower the RMSD value, the better the model was docked to the target structure [17,18].Fig. 2 showed that the RMSD value was 1.74 Å with a grid box size of 50 x 50 x 50 and coordinates x, y, and z (15,070,-2,582,-22,842).This implied that the molecular docking method carried out met the qualifications and showed the good quality of bond pose reproduction.

Fig. 2: Conformation overlay of native ligand validation result (blue) with natural ligan crystallography result (green)
In addition, the validation results also analyzed the active site of the amino acid residue of the protein that binds to the native ligand.Fig. 3 showed that the amino acid residues responsible for the binding of native ligands at the ACE binding sites were HIS513, GLU411, GLN281, HIS383, GLU384, HIS353, LYS511, HIS387, PHE457, TYR520, TYR523, ASP415, and VAL380.HIS513, GLU411, and GLN281 form hydrogen bond interactions.HIS383, GLU384, HIS353, LYS511, HIS387, PHE457, TYR520, TYR523, ASP415, and VAL380 form hydrophobic interactions.These amino acid residues are the amino acid residues that form the active site of ACE, so the existence of interactions with these amino acids is important when determining the antihypertensive activity of a compound [7].

Fig. 3: Interaction between native ligand and ACE
There were three parameters considered to determine the affinity of the test compound for the receptor, namely the bond energy ( ∆G), the inhibition constant (Ki), and the interaction with amino acid residues.Bond energy indicates the affinity between eucalyptol and the enzyme, the smaller the bond energy obtained, the more stable the bond formed [19].The ΔG value was directly proportional to the Ki value, the Ki value gave an idea of the ability of a compound to inhibit an enzyme.The smaller the Ki value, the compound had pharmacological capabilities in smaller doses [20].Eucalyptol had a bond energy value (∆G) of-6.40 kcal/mol with an inhibition constant of 20.82 µM.Interaction with amino acid residues indicated the presence of hydrogen bonds with HIS513 and HIS353 and hydrophobic interactions with the same amino acid residues of HIS383 and TYR523 as the native ligand.The functional groups of eucalyptol and captopril have similar hydrophobic interactions and hydrogen bonds to ACE amino acid residues.In addition, both have similar binding energies and inhibition constants (table 2).Based on the bond energy value, it showed that the eucalyptol had potential activity as an antihypertensive because it has an affinity and forms hydrogen bonds with the ACE.Overall, the molecular docking parameters of plant compounds of E. globulus can be seen in table 2. The visualization of molecular docking of captopril and tested compounds showed hydrogen bond and hydrophobic interaction between amino acid residues and the tested compounds.amino acid residues on the ACE active site, with captopril as the comparator drug, were 1,8-cineole, carvyl acetate, kaempferol, cubenol, taxifolin, and alpha-terpineol.Meanwhile, other compounds did not interact with important amino acid residues on the ACE active site because they bind to the other side, in contrast to captopril.Captopril interacted at the hydroxyl group with HIS513, HIS353, GLN128, LYS511, and TYR520, forming hydrogen bonds and forming hydrophobic interactions with ALA354, PHE457, and TYR523.Eucalyptol has the same 4 amino acid residues as captopril, namely HIS513, HIS353, TYR523, and ALA354.The carvyl acetate has the same 5 amino acid residues as captopril, namely TYR523, HIS353, HIS513, TYR520, and PHE457.
Among all the compounds, alpha-terpineol, carvyl acetate, and kaempferol had more bonds with the same amino acid residues as captopril, but these two compounds had bond energy affinity values and inhibition constants that were much different from captopril.Whereas 1,8-cineole (eucalyptol) and cubenol, which had almost the same binding energy affinity and inhibition constant values as captopril had interactions with amino acid residues that were similar to captopril.The toxicity profile of the 1,8-cineole based on AMES and TD50 tests showed that the compound was not mutagenic and not carcinogenic.The bond that occurs between eucalyptol and the ACE receptor can cause a decrease in blood pressure [21,22].
Molecular docking of the eucalyptol (1,8-cineole) was carried out using Autodock 4.2 program, with the coordinates of the interaction site being set the same as the coordinates of the native ligand on the ACE.The visualization of the molecular docking process between eucalyptol and ACE can be seen in fig. 4.  3 showed the ADMET analysis results for 62 compounds, only 2,6-dimethylocta-1,5,7-trien-3-ol, ellagic acid, cis-β-ocimene, cyclohexanol 2-methylene-5-(1-methyl ethenyl)-, exo-2hydroxycineole, isobornyl formate, limonene, naringenin, terpinene-4-ol, terpinolene, trans-carveol, α-guaiene, α-terpineol acetate, and γ-terpinene were only 15 mutagenic compounds.Predictive results for HIA (%, Human Intestinal Absorption), Caco2 (10 -6 cm/s) Caco-2 cell permeability assays to measure drug absorption, PPB (%) assays determine free drug concentration (fraction unbound) by evaluating affinity to plasma proteins, such as serum albumin, using plasma from treated animals, and BBB (blood-brain barrier) lets some substances, such as water, oxygen, carbon dioxide, and general anesthetics, pass into the brain.It also keeps out bacteria and other substances, such as many anticancer drugs that gave negative results of all tested compounds and gave reasonable predictions [12,23,24].All the test compounds met the requirements and could be used as oral drug candidates and could be further investigated for their pharmacokinetic and toxicity profiles.

CONCLUSION
The eucalyptol has potential activity as an antihypertensive because it has an affinity with a bond energy value (∆G) of -6.40 kcal/mol and an inhibition constant of 20.82 µM and has hydrogen bonding interactions with HIS513 and HIS353, and hydrophobic interactions with HIS383 and TYR523, so that they can inhibit ACE and can cause a decrease in blood pressure.

I
In nt te er rn na at ti io on na al l J Jo ou ur rn na al l o of f A Ap pp pl li ie ed d P Ph ha ar rm ma ac ce eu ut ti ic cs s 3 rd Bandung International Teleconference on Pharmacy, Indonesia |135 2017 software, which was set to view quality for publication.Then the water molecules in the structure were removed to simplify energy calculations when the simulation was carried out.The presence of water molecules in the structure causes the program to be unable to place the ligands correctly [14].The native ligand inhibitor of the ACE is (2S)-3-(4-hydroxyphenyl)-2-[[(2R)-2-[[hydroxy[(1R)2phenyl 1phenylmethoxycarbonyl aminoethyl] phosphoryl] methyl]-3-(3-phenyl-1,2-oxazol-5-yl) propanoyl] amino] propanoic acid was separated from the receptor structure.The structure of the ACE and native ligand can be seen in fig. 1.

R. Mustarichie et al. Int J App Pharm, Vol 15, Special Issue 2, 2023, 134-140 rd Bandung International Teleconference on Pharmacy, Indonesia |137Table 2 : Molecular docking of tested compounds of E. globulus to ACE
The tested compound must contain at least one amino acid residue that were the same as the native ligand amino acid residues so that it could be concluded that the tested compound has the potential to bind to ACE.The tested compounds that interact with important R.