INTRODUCTION:

Angiotensin I Converting Enzyme (ACE) or dipeptidyl carboxypeptidase plays a vital role in blood pressure homeostasis by hydrolyzing angiotensin I. The inactive peptide released after angiotensin cleavage by renin is converted into angiotensin II¹.

Angiotensin II is a ligand in the RAAS system that contributes to the development of cardiovascular diseases, including hypertension. Angiotensin II receptor Type 1 (ATR1) is a vasoconstrictive peptide that is a crucial regulator of blood pressure and sodium retention by the kidneys². Patients with chronic kidney disease can only be treated with ACE inhibitors and Angiotensin II Receptor Blockers (ARB)³.

Carica papaya is a neutraceutical plant that has various pharmacological activations⁴. The papaya plant has the benefit of protecting against cardiovascular disease and preventing harm caused by free radicals. It also helps in the treatment of diabetes mellitus and the reduction of cholesterol levels. The Papaya plant's fruit, leaves, and seeds can provide antioxidant, antihypertensive, hypoglycemic, and hypolipidemic, which can prevent and treat metabolic syndrome⁵.

Papaya leaves have the potential to be antihypertensive after in vivo research in rats. Research conducted on hypertension model rats given papaya leaf extract and enalapril control showed ACE inhibition and normalization of baroreflex sensibility. Active compounds (ligands) of papaya leaves that have the potential as antihypertensives, such as caffeic acid, rutin, and luteolin⁶. However, research on the potential of papaya leaves as antihypertensive through inhibition of ACE and angiotensin II receptors in silico has yet to be conducted. This study aims to predict the mechanisms of action of the active compounds in papaya leaves as antihypertensive agents through ACE and AT1R inhibition in silico.

MATERIALS AND METHODS:

Design, Time, and Place of Study:

The study uses computational design molecular docking with 3D structures of the target proteins ACE (code 1O86) and AT1R (code 4ZUD) in papaya leaves (Carica papaya). The docking process was carried out using AutoDock Tools. Active compounds papaya leaves were obtained from the dr. Duke's website and literature studies were screened that have potential as antihypertensives using the online PASS website. Physicochemical predictions and pharmacokinetics predictions were conducted using the SwissADME and pkCSM websites. The research was conducted in August 2023 at the Faculty of Medicine, University of Islam Malang

Tools and Materials:

The application used for docking is autodocktools version 1.5.7 (http://vina.scripps.edu/). The application for preparation and visualization 2D is Discovery Studio Visualizer v21.1.0.20298 (https://discover.3ds.com/discovery-studio-visualizer) and 3D is Chimera 1.17.3 (https://www.cgl.ucsf.edu/chimera/). Active compounds were found through website https://phytochem.nal.usda.gov/. The structure of the active compound of papaya leaves was downloaded through website http://pubchem.ncbinlm.nih.gov/. Screening of papaya leaf active compounds used website https://www.way2drug.com/passonline/. Target proteins ACE and AT1R bound with native ligands or drugs control were downloaded from PDB website http://rcsb.org. pkCSM website (https://biosig.lab.uq.edu.au/pkcsm) and SwissADME website (http://www.swissadme.ch/)

Screening of Antihypertensive Activity Potential:

31 active compounds papaya leaves were screened for the potential antihypertensive activity using PASS Online (Prediction of Activity Spectra for Substances). PASS Online is based on an analysis that contains information about the structure and biological activity of organic compounds that have antihypertensive potential Pa (Probability to be Active). The Pa value chosen is > 0.5. Pa value of 0.5 < Pa < 0.7 means that the active compound has the potential to be an active compound to be developed as a drug and Pa > 7 means potential to be an active compound and is similar to a medicinal compound⁷.

Target Protein and Ligand Preparation:

ACE and AT1R target proteins were prepared before docking by removing water molecules and interfering ligands that were not docked using application Discovery Studio Visualizer and saved in PDB format. After clean, insert the ligand into the autodock tools to give it a charge and hydrogen polar only. Attached native ligand has been separated from the target protein and cleaned from water and interfering ligands using the Discovery Studio Visualizer application and saved in PDB format. Ligand preparation is carried out to provide charge and provide all hydrogen using Autodock tools software⁸.

Validation of Molecular Docking:

The validation process uses the native ligand of the target protein and then redocking. Validation is done with a good binding affinity value compared to the native ligand. Validation is done by looking at the RMSD value between the native ligand and the test ligand before docking and after docking using the pymol 2.5.4 application (https://pymol.org/2/)⁹. A good RMSD value is <3.0 Å. Validation is also done by comparing the overlay between native ligands before docking and after docking. RMSD has three classifications for docking. First, the best classification is ≤2 Å, acceptable classification is between 2 Å and 3 Å, and bad classification is ≥ 3Å¹⁰.

Molecular Docking of Carica papaya Leaf Active Compounds to ACE and ATR1:

Docking is done by preparation of target protein, native ligand, and active compound. Preparation is done by cleaning water molecules and interfering ligands that can interfere with the docking process. Preparation is also done by adding hydrogen molecules and charges to the receptor and ligand. After that, we set the grid box. The grid box is adjusted to the size of the target protein on the native ligand. The grid box ACE (code 1O86) size x 28, size y 38, size z 44, X center 40.935 (Å), Y center 32.593 (Å), Z center 47.285 (Å).The grid box ATR1(code 4ZUD) size x 40, size y 32, size z 34, X center -41.038 (Å), Y center 63.098 (Å), Z center 28.373 (Å).After setting the grid box, the docking process was carried out using the autodock4 system autodocktools application with docking 100 repetitions. The docking result of the target protein with the ligand is obtained in .dlg format. After docking, the last process is conformational analysis on the best run on autodocktools to visualize amino acid residues in drug discovery studio application.

Data Analysis:

The docking result data analyzed are free binding energy (ΔG), inhibition constant (Ki), and amino acid residue. Free binding energy (ΔG) is an indicator of the affinity of the active compound to the target protein and is directly proportional to the Ki value. The similarity of amino acid residues with the native ligand indicates the same binding site as the control drug on the target protein.

ADMET Prediction and Physicochemical Properties:

ADMET (absorption, distribution, metabolism, excretion and toxicity) predictions are carried out using pkCSM website. pkCSM and SwissADME using SMILES (Simplified Molecular Input Line Entry System) from Pubchem¹¹. Physicochemical was carried out using SwissADME website and paste active compound SMILES in "Provide a SMILES string" then clicking ADMET¹².

RESULTS:

Molecular Docking Results:

Redocking result of ACE protein with native ligand Lisinopril got an RMSD 1.38 Å. Redocking result of AT1R protein with native ligand Olmesartan got an RMSD 2.71 Å. RMSD ≤ 3Å then included acceptable and valid¹².

Figure 1. Visualization of native Lisinopril (left) and Olmesartan (rihgt) ligand before docking in green and after docking in blue.

Docking Results of Papaya (Carica papaya) Leaf Active Compound and Lisinopril against ACE protein:

The free bond energy (∆G) of Lisinopril to ACE protein is -10.59 kcal/mol, Ki 0.017µ, and there are 10 amino acid residues TYR523, LYS 511,VAL380, HIS 513, GLU 384, HIS 383, GLU 376, HIS 353, GLU 162, and ALA354. 23 active compounds of papaya (Carica papaya) leaves have been docked and four active compounds have a free bond energy stronger than equal to -7 kcal/mol. The three active compounds are rutin, luteolin, epicatechin, and caffeic acid. Rutin has the most considerable affinity ∆G -9.19 kcal/mol, Ki 0.814 µM, and has a percentage of amino acid residue similarity 50% HIS513, GLU 384, HIS 353, LYS 511, and GLU 162. Luteolin has a ∆G -7.67 kcal/mol, Ki 2.4 µM, and a percentage of amino acid residue similarity 50%HIS 513, GLU 384,HIS 353, TYR 523, and HIS 383. Epicatechin has a ∆G -7.61, Ki 2.64 µM, and a percentage of amino acid residue similarity 50% HIS 513, GLU 384, TYR 523, HIS 383, and HIS 353. Caffeic acid has a ∆G -7.01 kcal/mol, Ki 7.31 µM, and a percentage of amino acid residue similarity 70% LYS 511, ALA 354, HIS 353, GLU 162, HIS 353,VAL 380, and ALA 354.

Docking Results of Papaya (Carica papaya) Leaf Active Compound and Olmesartan against Angiotensin II Receptor Type 1 (ATR1) Protein:

The result of free binding energy (∆G) of Olmesartan to ATR1 protein is -9.73 kcal/mol, Ki 0.073 µM, and there are ten amino acid residues TYR 292, ARG 167, SER 109, TYR 35, ILE 288, LEU 112, VAL 108, TYR 92, TYR 87, TRP 84. 23 active compounds of papaya (Carica papaya) leaves that were docked, the compound carpaine was found to have the lowest ∆G close to the native ligand. Carpaine has ∆G -7.98 kcal/mol, Ki 1.42 µM and with a percentage of amino acid residue bond similarity 20% ILE 288 and TYR 92. Rutin has a ∆G -6.86, Ki 9.4 µM, and a percentage amino acid residues 70% TYR 292, ILE 288, VAL 108, TYR 92, TRP 84, ARG 167, and TYR 35.

Figure 2. Visualization 2D of ACE interaction with Rutin (left) and AT1R interaction with Carpaine (right).

Figure 3. Visualization 3D of ACE interaction with Rutin (left) and AT1R interaction with Carpaine (right).

Table 1: Results of Interaction of Papaya Leaf Compounds with ACE Protein

Ligand	ΔG kkal/mol	Ki (µM)	Presentation of Amino Acid equations
Lisinopril (Native ligand)	-10.59	0.017	10/10 (100%)
Rutin	-9.19	0.184	5/10 (50%)
Luteolin	-7.67	2.4	5/10 (50%)
Epicatechin	-7.61	2.64	5/10 (50%)
Caffeic acid	-7.01	7.31	7/10 (70%)

Note: Docking results above -7 kcal/mol are not displayed in the table.

Table 2: Results of Interaction of Papaya Leaf Compounds with AT1R Protein

Ligand	ΔG kkal/mol	Ki (µM)	Presentation of Amino Acid equations
Olmesartan (Native ligand)	-9.73	0.073	10/10 (100%)
Carpaine	-7.98	1.42	2/10 (20%)
Rutin	-6.86	9.4	7/10 (70%)

Note: Docking results displayed are close to -7 kcal/mol.

Results Physicochemical Properties of Active Compounds of Papaya Leaf (Carica papaya):

Prediction of physicochemical properties five active compounds that had the best affinity after docking. The results of physicochemical properties with Lipinski indicators in table 3 show that the active compounds caffeic acid, carpaine, epicatechin, and luteolin meet the five Lipinski criteria, but rutin does not meet the Lipinski rule. Lipinski's rule is used to determine the solubility of active compounds. The Lipinski rules were log P, molecular weight, HBA, and HBD. Carpaine, epicatechin, and caffeic acid have good solubility through the lipid bilayer membrane and are widely distributed in the body because it is seen from the Log P value> 0. The active compounds rutin and luteolin have poor solubility because they have negative Log P values. Luteolin has a Log P value of -0.03, and rutin has a Log P value of -3.89. The rutin compound has a low permeability value at the intestinal and blood-brain barrier because it has a molecular weight value of 610.52 g/mol. Rutin is more challenging to penetrate the cell membrane.

Table 3: Physicochemical Properties of Active Compounds Papaya Leaf (Carica papaya)

Ligand	Chemical Formula	Log P	Molecular weight (g/mol)	HBA	HBD	Lipinski Rules
Caffeic acid	C₉H₈O₄	0.7	180.16	4	3	Yes
Carpaine	C₂₈H₅₀N₂O₄	3.75	478.71	6	2	Yes
Epicatechin	C₁₅H₁₄O₆	0.24	290.27	6	5	Yes
Luteolin	C₁₅H₁₀O₆	-0.03	286.24	6	4	Yes
Rutin	C₂₇H₃₀O₁₆	-3.89	610.52	16	10	No

Pharmacokinetics Properties Active Compounds of Papaya Leaf (Carica papaya):

ADMET prediction was performed on the five best active compounds using the pkCSM. Carpaine has the best intestinal absorption value of 92.27%. Active compounds are distributed in the high body based on the VDSS value. Rutin is the active compound distributed in the highest tissue than in the plasma. Active compounds that not penetrate to Blood-Brain Barrier (BBB) is rutin and epicatechin. Active compounds that do not affect CYP metabolism are rutin, luteolin, epicatechin, and caffeic acid. Carpaine affects CYP 3A4 metabolism. The active compounds carpaine, rutin, luteolin, epicatechin, and caffeic acid not toxicity effects on hepatotoxicity and AMES.

Table 4: Pharmacokinetics Properties of Active Compounds Papaya Leaf (Carica papaya)

Ligand	Absorption	Distribution		Metabolism				Excretion	Toxicity
Ligand	Intestinal Absorption (%)	Vdss (Log L/Kg)	BBB (log BB)	CYP 2D6 substrat	CYP 3A4 substrat	CYP 2D6 inhibitor	CYP 3A4 inhibitor	Total Clearance (log ml/min/kg)	Hepatotoksik	AMES
Caffeic acid	69.407	-1.098	-0.647	No	No	No	No	0.508	No	No
Carpaine	92.276	0.812	-0.355	No	Yes	No	No	0.897	No	No
Epicatechin	68.829	1.027	-1.054	No	No	No	No	0.183	No	No
Luteolin	81.13	1.153	-0.907	No	No	No	No	0.495	No	No
Rutin	23.446	1.663	-1.899	No	No	No	No	-0.369	No	No

DISCUSSION:

Affinity of Active Compounds Papaya Leaf (Carica papaya) to ACE and ATR1:

Docking results of papaya leaf active compounds on ACE protein in table 1 show that the free bond energy value of active compounds is weaker than native ligand or Lisinopril drug control. Rutin has the lowest free binding energy (∆G) value and the lowest inhibition constant (Ki) value on ACE protein compared to other active compounds. The ∆G and Ki values of the active compounds are directly proportional to each other. The four best active compounds include rutin ∆G -9.19 kcal/mol, Ki 0.184 uM; luteolin ∆G -7.67 kcal/mol, Ki 2.4 uM; epicatechin ∆G -7.61 kcal/mol, Ki 2.64 uM; and caffeic acid ∆G -7.01 kcal/mol, Ki 7.31 uM.

The docking results of 23 active papaya leaf compounds on the ATR1 protein in table 2 show that the free bond energy value of active compounds is weaker than native ligand or Olmesartan drug control. Carpaine is an active compound that lowest ∆G and Ki on the ATR1. The two best active compounds that potentially inhibit ATR1 are carpaine ∆G -7.98 kcal/mol, Ki 1.42 uM, and rutin ∆G -6.86 kcal/mol, Ki 9.4 uM. Carpaine has a low amino acid residue similarity of 20% compared to Olmesartan, so it has a weak potential to inhibit AT1R.

Active Compounds of Papaya Leaf:

Research conducted by Brasil et al. stated that the active compounds of Carica papaya have an inhibitory effect on ACE in vivo. The in vivo study was conducted on hypertensive rats treated with Enalapril and given methanol extract from Carica papaya leaves¹⁰. Research conducted by Guerrero et al. The antihypertensive effect of rutin was determined by estimating the inhibitory effect on ACE activity with in vitro studies. Rutin is an active compound of the flavonoid class. Rutin has pharmacological effects such as antioxidant, antidiabetic, antihypertensive, cardioprotection, and anti-inflammation. Rutin has a Ki value 0.184 µM in inhibiting ACE. In another study on inhibiting ACE activity in vitro had an IC-50 of 64 μM. IC-50 is concentration of a compound that inhibited ACE activity by 50%^13,14. In addition, studies conducted in rats showed that rutin can reduce ACE expression, which works similarly to lisinopril as an ACE inhibitor¹⁵. Rutin has a high amino acid residue similarity of 70% with the control drug Olmesartan but has low affinity so it has weak potential in inhibiting AT1R. Carpaine is an alkaloid group compound. Carpaine is recognized for its antiviral properties against dengue, as well as its antiplasmodium, antitumor, antihelminthic, and anti-inflammatory effects¹⁶. Carpaine has a low similarity in amino acid residues, namely 20% compared to Olmesartan, so it has weak potential in inhibiting AT1R. Luteolin is an active compound of the flavonoid group. Ki value of luteolin in inhibiting ACE activity is 2,4µM. Luteolin has high activity as an ACE inhibitor with an IC50 value of 23 uM in a study conducted using the fluorimetric method¹³. Luteolin can reduce blood pressure and aortic wall thickening in vivo¹⁷. Caffeic acid is an active compound of the carbocyclic acid group. In vivo studies of caffeic acid in rats showed inhibition of ACE inhibitors^6,18. Caffeic acid also has pharmacological effects as an antioxidant, immunomodulator, neuroprotective, anti-anxiolytic, antiproliferative, and anti-inflammatory activity. Epicatechin is an active compound of the flavonoid class. Epicatechin inhibits ACE activity based on research conducted in vivo in rabbits¹⁷. Epicatechin also has biological effects such as cardioprotective, antidiabetic, hepatoprotective, anti-inflammatory, antithrombotic, anticoagulant, antifibrotic, and antiviral¹⁸.

Physicochemical Prediction of Papaya Leaf Active Compounds that Potentially Inhibit ACE and ATR1:

Table 3 physicochemical tests are needed to determine the ability to absorb and permeability. The Lipinski rule used is that the H-Bond Donors (HBD) value is not more than equal to five, the H-Bond Acceptors (HBA) value is not more than equal to 10, the molecular weight is not more than equal to 500 daltons or g/mol, and Log P (Clog P) is not more than equal to five¹⁹. The active compounds with the best solubility are caffeic acid, carpaine, and epicatechin based on molecular weight, log P, HBA, and HBD. Caffeic acid has a molecular weight of 180.16 g/mol; log P 0.7; HBA 4 and HBD 3. Carpaine has a molecular weight of 478.71 g/mol, Log P 3.75, HBA 6, and HBD 2. Epicatechin has a molecular weight of 290.27; log P 0.24; HBA 6; HBD 5. Caffeic acid, carpaine, and epicatechin have good permeability in the digestive tract and lipid bilayer membrane, so that they can be well distributed in the body. Rutin has a molecular weight of 610.52 g/mol, HBA 16, and HBD 10, so it does not fulfill Lipinski's rule. Molecular weight determines the permeability of ligands in the digestive tract and the Blood-Brain Barrier (BBB). Molecular weight >500 daltons or g/mol has low permeability⁸. Excessive amounts of HBD and HBA impair permeability across the lipid bilayer membrane²⁰. Luteolin has a Log P value of -0.03, so it cannot pass through the lipid bilayer membrane. Therefore, the active compounds with high solubility are carpaine, caffeic acid, and epicatechin.

Pharmacokinetics Prediction of Papaya Leaf Active Compounds that Potentially Inhibit ACE and ATR1:

Pharmacokinetic-based predictions of the five best active compounds using the pkCSM website are shown in table 4. The potential of an active compound for optimal binding to therapeutic targets is essential to ensure the active compound can reach the target site in sufficient concentration to produce physiological effects safely²¹. Based on absorption indicators, carpaine and luteolin have absorption values >80% in the intestine. Good absorption has a role in increasing the concentration of active compounds in the plasma at the target protein. An active compound must have an intestinal human absorption rate of >30%²². Absorption in the human intestine is good if it has a presentation of > 80%⁸.

Based on distribution indicators, epicatechin and rutin can be freely distributed in the blood and cannot penetrate the BBB. VDSS is volume of active compounds that can be distributed in blood plasma evenly. The higher the Vdss, the more active compounds are distributed in tissues rather than plasma. A good VDSS (log L/kg) value is ≥ -0.15. The vdss value is high if > 0.45 Log L/kg and low if < -0.15 Log L/kg²². In addition to the VDSS value, the distribution is also influenced by the ability of the active compound not to penetrate the BBB. Permeability at the BBB refers to the capacity of active compounds to cross the blood-brain barrier. Active compounds with a logBB of less than -1 are unable to cross the blood-brain barrier, while those with a logBB greater than 0.3 can penetrate the blood-brain barrier²³.

Based on metabolic indicators, carpaine binds to the substrate metabolism enzyme CYP3A4. Cytochrome P450 (CYP450) is a detoxification enzyme present in the hepar. In general, cytochrome P450 is involved in drug metabolism. Inhibitors of cytochrome P450 can affect the pharmacokinetics of drugs. Cytochrome P450 oxidizes xenobiotics or compounds foreign to the body so that they can be excreted. Inhibitors of CYP2D6 and CYP3A4 enzymes can potentially interfere with drug metabolism²². CYP3A4 is an indicator of specific and cooperative substrate binding, leading to unwanted drug-drug interactions and toxic side effects. CYP3A4 can decrease the bioavailability and therapeutic efficiency of drugs through rapid degradation, and plasma drug levels may increase if CYP3A4 is inhibited. In addition to detoxification and clearance of xenobiotics, CYP3A4 exerts genotoxic effects through the activation of procarcinogens and contributes to adverse drug-drug interactions²⁴.

Based on excretion indicators, carpaine had the highest total clearance value of 0.897 log ml/min/kg. Excretion is seen through CLtot (total clearance) with units of Log ml/min/kg. A high CLtot indicates that the compound is excreted faster. Total clearance is a combination of hepatic clearance and renal clearance⁸. Based on toxicity indicators, caffeic acid, carpaine, epicatechin, luteolin, and rutin have no hepatotoxic effects and AMES. Hepatotoxicity is hepatic damage caused by exposure to compounds due to adverse compound reactions²⁵. Ames is a compound toxicity test with bacteria to assess mutagenic potential. If the compound has a positive Ames test, it has mutagenic properties that can become a cancer-causing carcinogen²³. The active compounds caffeic acid, carpaine, epicatechin, luteolin, and rutin do not cause carcinogenic cancer. Therefore, the active compounds that have good pharmacokinetics are carpaine and luteolin.

Molecular docking has an advantage as it can predict interactions between small molecules and proteins at the atomic level. It can identify the binding site of ligands on the target protein. Therefore, molecular docking plays a vital role in assessing the potential of active compounds in herbs for developing disease treatments²⁶. Molecular docking has limitations in determining the stability of affinity in active compounds. Further research can be conducted using Molecular Dynamics Simulations (MDS) to assess the stability of active compounds. MDS can accurately control conditions such as temperature and ions around peptides, as well as solvent properties such as volume, types of solvents (e.g., water, organic solvents), or ionic liquids²⁷. Molecular docking can be integrated with molecular dynamics simulations to examine the dynamic interactions within protein-ligand complexes. These simulations help in understanding the conformational changes that occur during ligand binding and the stability of the resulting complex²⁶. Furthermore, in vivo research on papaya leaves as an antihypertensive through the mechanism of AT1R inhibition has not yet been conducted. Therefore, this study needs to be continued in vivo to demonstrate that the active compounds in papaya leaf extract have a mechanism for inhibiting AT1R.

CONCLUSION:

The active compounds rutin, luteolin, epicatechin, and caffeic acid potentially inhibit ACE with weaker affinity than Lisinopril as a control drug. The active compounds carpaine and rutin have the potential to inhibit ATR1 but have a weaker affinity than Olmesartan as a control drug. Active compounds with the best solubility and meet the Lipinski rule criteria are caffeic acid, carpaine, and epicatechin, suitable for peroral consumption. The active compounds caffeic acid, carpaine, epicatechin, luteolin, and rutin can become drugs.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank the supervisor who has guided us from beginning to end. The Student Parents Association and the Faculty of Medicine of UNISMA, as well as research team friends who helped carry out this research.

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Received on 19.12.2023 Revised on 22.04.2024

Accepted on 16.07.2024 Published on 24.12.2024

Available online from December 27, 2024

Research J. Pharmacy and Technology. 2024;17(12):5908-5914.

DOI: 10.52711/0974-360X.2024.00896