1Doctoral Program of Pharmaceutical Sciences, Faculty of Pharmacy, Universitas Airlangga, Gedung Nanizar Zaman Joenoes Kampus C UNAIR Jalan Mulyorejo Surabaya, East Java, Indonesia, 60115.
2Department of Pharmaceutical Science, Faculty of Pharmacy, Universitas Airlangga, Gedung Nanizar Zaman Joenoes Kampus C UNAIR Jalan Mulyorejo Surabaya, East Java, Indonesia, 60115.
3Skin and Cosmetic Technology (SCT) Center of Excellent, Universitas Airlangga, Gedung Nanizar Zaman Joenoes Kampus C UNAIR Jalan Mulyorejo Surabaya, East Java, Indonesia, 60115.
4Faculty of Pharmacy, Institut Ilmu Kesehatan Bhakti Wiyata, Jalan KH Wahid Hasyim 65 Kediri,
East Java, Indonesia, 64117.
*Corresponding Author E-mail: tristiana-e-m@ff.unair.ac.id
ABSTRACT:
Butterfly pea (Clitoria ternatea) is a plant that has been widely studied as an antioxidant. Antioxidants are compounds that are believed to have various activities, one of which is that they can be used as whitening agents on the skin. The antioxidant ability of water extract of butterfly pea flower is thought to come from the anthocyanin component in the flowers. The objective of this research is to determine the inhibitory activity of the enzyme tyrosinase from 5 of the most abundant anthocyanins in water extracts of butterfly pea flowers. The method of this study, docking of test compounds against target receptors, was carried out using the Autodock Tools and Autodock 4.2 programs. From the results of molecular Docking, it was found that the five most abundant anthocyanins from butterfly pea flowers were Cyanidin 3-(p-coumaroyl)-glucoside, Cyanidin-3-(p-coumaroyl)-rutinoside-5-glucoside, Delphinidin-3-O-(6- p- coumaroyl)glucoside, Delphinidin-3-(p-coumaroyl)-rutinoside-5-glucoside, and Delphinidin 3-O-glucoside have the same activity as hydroquinone as anti-melanogenesis when viewed from the ΔG, Ki, and residue similarity values amino acids that interact with the ligand. However, Delphinidin-3-O-(6-p-coumaroyl) glucoside has the lowest ΔG value of -9.23 kcal/mol with a Ki of 170.82nM compared to hydroquinone and other test compounds.
KEYWORDS: hydroquinone, telang, tyrosinase, whitening.
INTRODUCTION:
One of the most popular types of cosmetic products is skin-lightening products. Skin-lightening products usually contain ingredients that inhibit melanin formation. Generally, the ingredients used in skin-lightening products are tyrosinase inhibitors, plant extracts and antioxidants1,2.
Melanin is a natural pigment found in the body in the epidermis. Melanocytes produce melanin through a process called melanogenesis. Melanin functions to protect the skin from harmful ultraviolet radiation from the sun. Even though melanin has a crucial function, excessive melanin production can cause skin hyperpigmentation. The tyrosinase enzyme plays an important role in melanin production. So, increasing the activity of the tyrosinase enzyme can result in hyperpigmentation. Exposure to UV rays, some chemical compounds, and certain diseases can also cause hyperpigmentation3,4.
One of the active ingredients that is widely used to treat melanogenesis is Hydroquinone because of its ability to suppress melanocytes to inhibit melanin formation. The use of Hydroquinone for anti-melanogenesis is limited to a doctor's prescription because it has various side effects, including increasing the risk of cancer, irritating the skin, damaging DNA, increasing oxidative stress and causing cell apoptosis5,6. Ingredients derived from plants have received a lot of attention in treating hyperpigmentation because they are considered to have a milder, safer, healthier and more cost-effective effect. Several plants, plant extracts and phytochemicals with multiple mechanisms of action have been reported as effective depigmentation agents with less severe side effects7,8,9.
The plant known as butterfly pea blossom (Clitoria ternatea L.) is a member of the legume family of Fabaceae. This plant is a wild plant that usually grows in home gardens or around plantations or rice fields. Among Indonesian people, butterfly pea flowers are widely used as a natural dye, processed into drinks, as an ornamental plant, and also used as herbal medicine10. Anthocyanins, found in butterfly pea blossoms, are naturally occurring antioxidants that slow down aging7. Anthocyanins are blue-purple-red-orange colored plant flavonoids11.
The content of anthocyanin compounds is the cause of the distinctive bluish-purple colour of butterfly pea flowers. Apart from that, butterfly pea flowers contain alkaloids, flavonoids, saponins, quinones, polyphenolics and triterpenoids. Anthocyanin is one of the flavonoid compounds that has the highest and most stable antioxidant activity because it is polyacylated (has > 2 native groups)12,13. Research conducted by Karunarathne et al., 201914 proved that anthocyanin in compounds have the potential to have anti-melanogenesis activity. The most anthocyanin content in butterfly pea flowers is the group Cyanidin 3-(p-coumaroyl)-glucoside, Cyanidin-3-(p-coumaroyl)-rutinoside-5- glucoside, Delphinidin-3-O-(6-p-coumaroyl) glucoside, Delphinidin-3-(p-coumaroyl)-rutinoside-5-glucoside, and Delphinidin 3-O-glucoside)15.
Molecular docking can be used to determine the activity of the five anthocyanin compounds. Molecular docking is an essential tool in drug discovery16. Docking is a crucial tool for lead optimization. It can virtually screen various compounds, rate the results, and suggest structural theories about how the ligands block the target17.
In this research, molecular docking of five anthocyanin compounds found in butterfly pea flowers was carried out on the inhibitory activity of the tyrosinase enzyme compared with hydroquinone as control. The receptor was used is 4P6R. The 4P6R receptor is a tyrosinase enzyme with good resolution originating from Bacillus megaterium. 4P6R shows tyrosinase activity towards L-tyrosine and L-Dopa18.
The tools used in this research include computer hardware with AMD 3020e Processor specifications with Radeon Graphics (2 CPUs), ~1.2GHz, 4 GB RAM, and Windows 10 operating system software. The software used is ChemDraw, AutodockTools 1.5. 6, Discovery Studio Visualizer, ProTox, AdmetLab and Lipinski's Rules of Five.
Toxicity and Lipinski’s Rules of Five Prediction:
The 3D structure of the tyrosinase enzyme was downloaded from the Protein Data Bank (PDB ID: 4P6R) and prepared in AutodoksTools 1.5.6. The conformations of the five test compounds are Cyanidin 3-(p-coumaroyl)-glucoside, Cyanidin-3-(p-coumaroyl)-rutinoside-5-glucoside, Delphinidin-3-O-(6-p-coumaroyl) glucoside, Delphinidin-3 -(p- coumaroyl)-rutinoside-5-glucoside, and Delphinidin 3-O-glucoside were obtained from PubChem with 3D display.
The five compounds were prepared using ChemDraw with the output file extension (*.pdb). The output results were visualized using Discovery Studio Visualizer.
The validation method used in the docking process in this research is the redocking method, with pose selection carried out on the receptor's active site that binds to the ligand19. The value obtained is the Root Mean Square Deviation (RMSD). Method validation is said to be valid if an RSMD value ≤ 2Å.
Test compounds were divided into target receptors using the AutodockTools and Autodock 4.2 programs. The first stage is the preparation of ligands and receptors using the AutodockTools program. The second stage is determining the grid box using the AutodockTools program to determine the receptor binding site. The third stage starts the docking process with the Autodock 4.2 program and then runs it. Two processes will be carried out during running, namely autogrid and autodock. When running autogrid, a *.glg file will be generated, and when running autodock, a *.dlg file will be generated.
The Discovery Studio Visualizer software visualised the docking results by viewing the bonds formed in the ligand and receptor interactions in 2D and 3D.
The validation of the docking method carried out in the docking process this time was the redocking method with pose selection carried out on the active site of the receptor that binds to the ligand19. The purpose of docking is to confirm the simulation's dependability. By removing the protein from the native ligand attached to the receptor, validation was completed. Redocking was carried out to ascertain the RMSD value and validate the anticipated inhibitor structure and binding energy. The degree of variation between crystallographic ligand results at the same binding site and experimental ligand docking results is displayed by RMSD. A larger deviation denotes a more substantial inaccuracy in the prediction of ligand-receptor interactions and the greater the RMSD value. If the RMSD value of a molecular docking approach is less than 2 Å, it is considered legitimate. In Figure 1, it can be seen that the validation results of docking of the tyrosinase enzyme receptor against the hydroquinone ligand with grid box sizes of 20, 20, 20 and number of points x, y, z (9.526, 9.941, 0.038), obtained an RMSD value of 0.29. The RMSD Value shows that the docking method used is valid and the parameter settings used meet the validation criteria.
Figure 1. Natural (grey) and crystallographic (red) ligand conformational re-docking results
Toxicity Prediction
Toxicity prediction using the ProTox II web program is shown in the following table:
|
No |
Compound |
Molecule Weight <500 g/mol |
Hydrogen Donor <5 |
Hydrogen Acceptor <10 |
Log P <5 |
Refractory Molar 40-130 |
|
1 |
Hydroquinone |
110 |
2 |
2 |
1,1 |
29,77 |
|
2 |
Cyanidin 3-(p-coumaroyl)-glucoside |
593 |
6 |
13 |
1,5 |
146,33 |
|
3 |
Cyanidin-3-(p-coumaroyl)-rutinoside-5-glucoside |
903 |
13 |
22 |
(-) 2,11 |
207,31 |
|
4 |
Delphinidin-3-O-(6-p-coumaroyl) glucoside |
611 |
9 |
14 |
1,87 |
149,13 |
|
5 |
Delphinidin-3-(p-coumaroyl)-rutinoside-5- glucoside |
911 |
14 |
23 |
(-) 2,22 |
208,88 |
|
6 |
Delphinidin 3-O-glucoside |
465 |
9 |
12 |
(-) 0,102 |
108,12 |
Table 2. Result of Toxicity Prediction
|
No |
Compound |
Toxicity Parameter |
||
|
Carcinogenicity |
Mutagenicity |
Skin sensitization |
||
|
1 |
Hydroquinone |
+ |
- |
+ |
|
2 |
Cyanidin 3-(p-coumaroyl)-glucoside |
- |
- |
- |
|
3 |
Cyanidin-3-(p-coumaroyl)-rutinoside-5-glucoside |
- |
- |
- |
|
4 |
Delphinidin-3-O-(6-p-coumaroyl) glucoside |
- |
- |
- |
|
5 |
Delphinidin-3-(p-coumaroyl)-rutinoside-5-glucoside |
- |
- |
- |
|
6 |
Delphinidin 3-O-glucoside |
- |
- |
- |
Table 3. Result of Molecular Docking
|
No |
Compound |
ΔG (kcal/mol) |
Ki |
Hydrophobic Bond |
Hydrogen Bond |
|
1 |
Hydroquinone |
-4,68 |
371,27 µM |
PRO90, LYS9, LEU14 |
GLU18, THR88, VAL7 |
|
2 |
Cyanidin 3-(p-coumaroyl)-glucoside |
-5,21 |
152,25 µM |
TYR5, VAL7 |
GLU86, VAL7, THR88, ARG6, |
|
3 |
Cyanidin-3-(p-coumaroyl)-rutinoside-5-glucoside |
-2,23 |
23,12 mM |
VAL7 |
GLU18, GLU86, VAL7, LYS4, LYS9, THR88 |
|
4 |
Delphinidin-3-O-(6-p-coumaroyl) glucoside |
-9,23 |
170,82 nM |
PRO90, LYS9, LEU14 |
GLU18, GLU86, VAL7, ASP21, THR88, LYS4 |
|
5 |
Delphinidin-3-(p-coumaroyl)-rutinoside-5- glucoside |
-3,03 |
5,98 mM |
LEU290, VAL7 |
ARG6, VAL7, LEU209 |
|
6 |
Delphinidin 3-O-glucoside |
-2,61 |
12,16 mM |
_ |
VAL7, ARG6, TYR5, GLU86 |
Figure 2. Visualization of molecular docking of Hydroquinone against tyrosinase enzyme. A: 2D; B: 3D.
Figure 3. Visualization of molecular docking of Cyanidin 3-(p-coumaroyl)-glucoside against tyrosinase enzyme. A: 2D; B: 3D.
Figure 4. Visualization of molecular docking of Cyanidin-3-(p-coumaroyl)-rutinoside-5-glucoside against tyrosinase enzyme. A: 2D; B: 3D.
Figure 5. Visualization of molecular docking of Delphinidin-3-O-(6-p-coumaroyl) glucoside against tyrosinase enzyme. A: 2D; B: 3D.
Figure 6. Visualization of molecular docking of Delphinidin-3-(p-coumaroyl)-rutinoside-5-glucoside against tyrosinase enzyme. A: 2D; B: 3D.
Figure 7. Visualization of molecular docking of Delphinidin 3-O-glucoside against tyrosinase enzyme. A: 2D; B: 3D.
Lipinski's Rules of Five evaluates the similarity of compounds with the characteristics of oral drugs that are biologically active in humans. This rule states that the molecule must obey the following components: <5 hydrogen bond donors; <10 hydrogen bond acceptors because a large number of hydrogen bonds will reduce the distribution of the molecule from the water-soluble phase to the passive diffusion permeable lipid bilayer membrane; Molecular weight < 500 Dalton because high molecular weight reduces the concentration of compounds on the surface of the intestinal epithelium, thereby reducing absorption; < 5log P (octanol-water partition coefficient) because high log P values can cause poor malabsorption20,21. Based on Table 1, the five compounds tested did not meet Lipinski's Rules of Five parameters, so they could not be used orally. However, it can be used topically to penetrate the membrane well.
Toxicity prediction was performed on 5 test compounds of anthocyanin in butterfly pea and hydroquinone as control positive in anti-melanogenesis. This test uses the ProTox II web program with carcinogenicity, mutagenicity, and skin sensitization parameters. Carcinogenicity prediction predicts compounds that can potentially cause cancer development. Mutagenicity predicts short-term reverse mutations in bacteria in detecting potential compounds that can cause genetic damage. Skin sensitizer prediction or skin sensitivity is used to determine the potential of a compound that can cause allergic reactions such as rashes, blisters, or itching when in direct contact with the skin 22. Based on this test, all of 5 test compounds are predicted to have a better level of toxicity than hydroquinone.
Molecular docking was carried out with the target enzyme tyrosinase, and the target protein used is 4P6R. The enzyme tyrosinase is a protein specific for melanogenesis activity, therefore, it is hoped that the test compound will bind to the receptor and can treat hyperpigmentation or aging diseases23. 4P6R protein was prepared using Autodock for water deletion. Removal of water molecules in protein because, during the docking process, it is possible that water will bind to the ligand to form hydrogen bonds and can disrupt the binding of the molecules during the docking process.
Molecular docking of the test compound on the tyrosinase enzyme receptor is carried out by looking at the results of the binding free energy (ΔG) and inhibition constant (Ki). Based on the docking results in Table 3, there are five compounds predicted to have the same activity as hydroquinone. However, one of the best compounds is Delphinidin-3-O-(6-p-coumaroyl) glucoside, which has the lowest free energy value of -9.23 kcal/mol with a Ki value of 170.82nM. The low ΔG value makes the complex conformation formed predicted to be more stable than other test compounds24.
Binding free energy (ΔG) shows how strong the bond between the receptor and the ligand is. Good affinity is in the form of an ΔG value that is increasingly negative (low). Low affinity indicates that the compound requires little energy when binding, so it can be said that low affinity indicates that the compound has greater potential to interact and form strong bonds with the target compound. The value of the inhibition constant (Ki) is directly proportional to the value of ΔG; the more negative the value of ΔG, the lower the value of the inhibition constant (Ki). The Ki value describes the binding affinity with a value in the form of the concentration required for a substance to reduce enzyme activity. The smaller the Ki value, the greater the affinity of the ligand with the receptor and the lower the compound concentration required to inhibit enzyme activity.
Discovery Studio Visualizer is used to visualize the ligand interactions with the target protein that result from docking. The outcomes of visualizing the ligand in two and three dimensions with the target protein are displayed in Figures 2 through 7 and are compiled in Table 3. The five compounds have the same residues as the hydroquinone ligand against target proteins. If there are many similar residues between the reference ligand and the test ligand, then the level of similarity in properties will be higher. From the docking score and similarity of the residues involved, the five compounds can be said to have anti-melanogenesis activity.
From the predictions Lipinski Rule of Five, it is known that the five types of anthocyanins in extract of Butterfly Pea flowers is thought to have no activity as an oral preparation but can be used as a topical preparation. From the results of toxicity tests on the skin, five types of anthocyanins are safer than Hydroquinone. Also, it has a smaller delta G value compared to Hydroquinone, so it is thought to have more potent and safer anti-melanogenesis activity compared to Hydroquinone.
Authors will include statement about no conflict of interest.
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Received on 24.02.2024 Revised on 13.08.2024 Accepted on 11.01.2025 Published on 02.05.2025 Available online from May 07, 2025 Research J. Pharmacy and Technology. 2025;18(5):2043-2048. DOI: 10.52711/0974-360X.2025.00292 © RJPT All right reserved
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