INTRODUCTION:

As part of the process of living, humans use O2 to oxidize biologic substrates for energy supply via aerobic survival. This process produces free radical forms of oxygen that are capable of damaging cells by the oxidation process of cell components and molecules known as reactive oxygen species (ROS). The cells are capable to maintain a redox state when low or moderate levels of ROS are produced, however, increased ROS would lead to oxidative stress and cellular damage.¹Antioxidant defense to counter ROS consists of the non-enzymatic and enzymatic pathways.

The non-enzymatic pathway involves molecules (i.e., retinol, ascorbic acid, tocopherol, etc) that directly quench ROS to prevent further oxidative damage. The enzymatic pathway mainly involved three main antioxidant enzymes: superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase. The enzymes work compartmental and are controlled genetically.^2–4

SOD is the first line in the enzymatic antioxidant protection framework. SOD works by changing anion superoxide as the strong initiator to the common chain reaction to a more stable compound like oxygen (O2) and hydrogen peroxide (H2O2). SOD level in the body is affected by numerous factors including age, gender, body mass index, alcohol consumption, menopause, drug consumption, transferrin level, ferritin, albumin, bilirubin, haptoglobin, total protein, uric acid, hemoglobin, and mean corpuscular volume of the erythrocyte.^5,6

There are three isoforms of SOD that have been found in humans, of which are the cytosolic CuZnSOD (SOD1), MnSOD (SOD2), and extracellular CuZnSOD (SOD3).⁷ All the SODs require metal for transition redox in its active site. SOD1 is mainly located in intracellular and functions as an antioxidant defense and mutation that has been linked to neurogenerative disease.^2,8 SOD3, one of the SOD isoforms, is found abundantly in extracellular locations and is expressed in cell type or tissue-dependent manner. SOD 3 has been hailed as the principal regulator of endothelium NO bioactivity through its O2•- scavenger activity. SOD3 has been suggested to play important roles in neurologic disorders, cardiovascular disorders, and cancer.^9–11Study also considered SOD3 as a good agent for the prevention or treatment of skin inflammation.¹¹ SOD2 is the only SOD isoform that is manganese-dependent and present in mitochondria. SOD2 is essentially for aerobic survival and the null homozygous mutation is known to be lethal.^12,13

The primary mechanism to eliminate H2O2 and lipid peroxides in the cytosol and mitochondria is catalyzed by GPx, which uses glutathione (GSH) to reduce H2O2 and hydroperoxides into water and alcohols, respectively. GPx, located in the cytosol and mitochondria, detoxifies H2O2 and hydroperoxides (ROOH) into H2O and alcohols (ROH), respectively.¹⁴ Different GPx isoforms have been identified in mammals.¹⁵ Although sharing the ability to reduce H2O2, isoforms differ in tissue expression and substrate requirement. This unique characteristic optimizes their antioxidant role.

Herbal extracts have been used in extensive fields of medicine for centuries. The most compelling effect of herbal medication is the compromise of the abundant improvement with minimum side effects. Centella asiatica, a well-known member of Umbelliferae family in herbal medicine, has shown some benefits in wound healing, memory improvement, headache, leprosy even in the treatment of renal failures.^16,17Centella asiatica also has the ability to counteract some oxidative stress activity.^18,19 In several studies, Centella asiatica significantly shows antioxidant activity in arsenic-induced oxidative stress, adriamycin-induced cardiomyopathy, and age-related neurological disorder.^20–22Centella asiatica had been identified as a potential effective antioxidant with comparable antioxidant activity to ascorbic acid and butylated hydroxytoluene.²³

The activity of Centella asiatica in oxidative stress is theorized to ranging from direct radical scavenging, ion chelating, enzyme activities, inhibiting lipid peroxidation, and preventive of DNA damage or protein modification. In silico method intends to predict the likelihood of target to yield approved drug thus capable for assessing Centella asiatica antioxidant properties. The selection of a good target is important for the discovery of an effective drug.^24,25 In silico methods have been developed and applied in bio-molecular and biotechnology studies for further development in pharmacology hypotheses. As a method, in silico studies had been a success in measuring the affinity of a new molecule to target and assessing physiochemical (absorption, distribution, metabolism, excretion) of new drugs.^26–29

This study specifically assessed the interaction of the active compound of Centella asiatica with SOD and GPx. This study used Autodock tools program to simulate binding between Centella asiatica and protein molecule SOD. Molecular docking is a computerized procedure for predicting non-covalent binding between macromolecule and ligand.

METHODS:

This study was conducted using software MarvinBean Suite^®, Open Babel^®, PyMOL^® verse 1.5.03 Open Source, Autodock 4.2^®, Chimera^®, JChem^® for Excel. Software was run in PC Intel^® core i7 3770 3,5 GHz, Quad Core-8 threaded, RAM 32 GB DDR3 10600.

Ligand and Protein Preparation:

Ligand for Centella asiatica derivatives was retrieved from Pubchem^® search engine. Meanwhile, the protein target in SOD was generated from three-dimensional structure modeling in .pdb format. This three-dimensional structure was obtained from www.pdb.org. Furthermore, OEChem^® V-2000 was used to optimize the three-dimensional structure.

Crystallography protein was downloaded from protein bank data http://www.rcsb.org/pdb/. For SOD, the crystal structure of human extracellular copper-zinc superoxide dismutase (2JLP) and the crystal structure of human glutathione peroxidase 5 (2I3Y) were used.^30,31 Chimera^® program was used for the preparation of the ligand. Protein 2JLP and 2I3Y were optimized through the addition of hydrogen or charge adjustment methods. Docking was conducted between ligand and protein with MOE 2020. The complex formed from the docking was analyzed. According to the interaction model from protein bank data, ligand co-crystal was selected as the crystallography ligand in the validation process.

Docking Validation:

Docking validation was conducted by using the re-docking method. After docking process, the ligand was saved then measured against the crystallography ligand. The measurement gave Root Mean Square Deviation (RMSD) in ligands which resulted in RMSD < 2, of which were eligible for the next process. Proteins from the docking process and derivatives from the ligand were put into MOE 2020 program. The results were saved in .csv format and .sdf format. The analytical process was based on binding energy, binding affinity, and interaction residue from molecular docking.

RESULTS AND DISCUSSION:

Parameters used in this molecular docking study were binding energy and binding affinity. Binding energy shows energy needed for the ligand to bind into the protein target. Lower binding energy shows more spontaneous and more strength of the binding. Binding energy was used as the primary parameter for determining the strongest bond formed amongst ligands. Binding affinity shows the degree of dissociation of ligand-protein target binds in pKi value.

Protein residue found in the study was used for identifying the location of hydrogen binding between ligand and protein target. Ligand binding to receptor binding site shows ligand capability as a protein enhancer. Location of receptor binding site and receptor active site of the target protein depicted in Table 1.

Table 1. Binding site of the protein target from literature.^32,33

Target Protein	Receptor Binding Site
Superoxide Dismutase (SOD)
Cu2+	47,49,64,121
Zn2+	64,72,81,84
Glutathione	81,84,86,89,92,96

Table 2. Binding energy and residue components of each ligand with 2JLP (SOD)

Molecules	∆G (kcal/mol)	pKi	H don & H acc
Asiatic acid	-8.8093	6.213	Gln118, Arg169
Asiaticoside	-10.2310	6.572	Glu158, Gln118
Batulinic acid	-7.8176	4.907	Arg169
Brahmol	-6.3866	5.504	Asp99, Asp99, Asp99, Arg109, Arg109
Centellasapogenol	-6.2375	4.513	Glu158
Isothankunik acid	-8.0146	4.831	Gln188
Madecassic acid	-9.0518	6.257	Arg169, Arg169, Gln118
Madasiatic acid	-8.6821	5.605	Tyr162
Terminolic acid	-8.4729	6.203	Gln118, Arg169

∆G (mean binding energy); pKi (binding affinity); H don (Hydrogen donor); H acc (Hydrogen Acceptor)

Figure 1. (A) 2 D model binding of ligand asiaticoside with 2JLP (SOD); (B) 3D model binding ligand of ligand asiaticoside with 2JLP (SOD)

Based on the data of ligand-2JLP binding depicted in Table 2, two bindings made by asiaticoside and madecassic acid were requiring lower energy amongst the ligands. Asiaticoside with binding energy -10.2310 kcal/mol and madecassic acid with binding energy -9.0518 kcal/mol. Based on protein residue, protein majorly bonds into Gln 118. Both asiaticoside and madecassic acid bond to Gln118.

Figure 2. (A) 2 D model binding of ligand madecassic acid with 2JLP (SOD);

(B) 3D model binding ligand of ligand madecassic acid with 2JLP (SOD)

Table 3. Binding energy and residue components of each ligand with 2I3Y (GPx)

Molecules	∆G (kcal/mol)	pKi	H don & H acc
Asiatic acid	-8.5552	7.988	Lys310, lys310
Asiaticoside	-9.8082	8.102	Glu239, Glu239, Glu244, Met278, Arg189, lys212
Batulinic acid	-7.3972	6.909	Glu185, Glu185, Lys256, Lys256
Brahmol	-6.4946	4.720	Lys256, Lys390
Centellasapogenol	-7.2673	4.799	Lys412, Lys412
Isothankunik acid	-8.4897	7.806	Arg224, Arg2244, Arg224, Lys310, Lys310
Madecassic acid	-8.1232	7.700	Asp178, Lys53, Gln182, Asn294
Madasiatic acid	-10.1232	8.650	Glu239, Glu185, Glu244, Arg224, Arg189, lys310
Terminolic acid	-8.0813	5.814	Arg224, Arg189

∆G (mean binding energy); pKi (binding affinity); H don (Hydrogen donor); H acc (Hydrogen Acceptor)

The result of docking study 2I3Y-ligand in Table 3 showed that madasiatic acid, asiaticoside, and asiatic acid performed with the lowest binding energy ligand, respectively -10.1232, -9.8082, and -8.5552 kcal/mol. Both madasiatic acid and asiaticoside had common binding residue of Arg189, Glu239, and Glu244. Meanwhile, madasiatic and asiatic acid had Lys310 common residue.

Figure 3. (A) 2 D model binding of ligand madasiatic acid with 2I3Y (GPx);

(B) 3D model binding ligand of ligand madasiatic with 2I3Y (GPx)

The docking in this study had proved the interaction between the active compound of the Centella asiatica with SOD (2JLP), with asiaticoside and madecassic acid showing the most significant interaction. The interaction virtue Centella asiatica antioxidant in-vivo activity in several studies. In the study by Yang et al. madecassic acid treatment increased the level of SOD in hypoxia-induced retinal microvascular endothelial.³⁴ Another study by Lee et al. asiatic acid in a titrated extract of Centella asiatica (TECA) had been known to inhibit the decreasing level of SOD3 in lung tissue of smokers. SOD3 is the major extracellular enzyme induced by antioxidants. Therefore,asiatic acid antioxidant ability might be due to SOD3.³⁵ However, there is no study discussing asiaticoside in-vivo experiments with SOD.

Our study found that madasiatic acid and asiaticoside bound significantly with 2I3Y (GPx). The previous study in modelled animal study had found that there was a significant increase of GPx in mice treated with 200 mg/kg CA.³⁶ Another study also found a significant GPx increase in hypoxia-induced mice after treatment with CA extract 300 mg/kg.³⁷

CONCLUSION:

In summary, this study had found that Centella asiatica active compound bound into 2JLP (SOD) and 2I3Y (GPx). Two ligands bind with significant binding energy, the asiaticoside and madecassic acid, with the binding energy of -10.2310 kcal/mol and -9.0518 kcal/mol, respectively. There was a significant GPx-ligand bind of madasiatic acid and asiaticoside, with the binding energy ligand of -10.1232 and -9.8082 kcal/mol, respectively. This study supports several in-vivo studies of madecassic acid interaction with SOD and GPx. However, further study is essential for assessing in-vivo experiments of another active compound of Centella asiatica.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 29.12.2021 Modified on 17.05.2022

Research J. Pharm. and Tech 2023; 16(1):399-403.

DOI: 10.52711/0974-360X.2023.00068