In Silico Docking of Phaleria Macrocarpa Bark Compounds Through Inflammation Pathway and its Cytotoxic Activities Against HCT116 Cell Line

Introduction

Colorectal cancer (CRC) is the one of the main reasons of most cancer-associated deaths within the international. CRC is accountable for greater than 600,000 deaths annually and occurrence charges are growing in most of the developing countries. Epidemiological and laboratory investigations advise that environmental elements consisting of western style nutritional behavior, tobacco-smoking, and absence of physical sports are taken into consideration as risks for CRC. Molecular pathobiology of CRC implicates pro-inflammatory situations to promote the tumor malignant progression, invasion, and metastasis. it’s far well known that sufferers with inflammatory bowel ailment are at higher threat of CRC. Many evidences exist reiterating the link between inflammation and CRC. infection includes interaction among numerous immune cells, inflammatory cells, chemokines, cytokines, and seasoned-inflammatory mediators, consisting of cyclooxygenase (COX) and lipoxygenase (LOX) pathways, which might also cause a signaling towards growth and proliferation cancer cell.

Phaleria macrocarpa (Scheff.) Boerl or Mahkota dewa in Indonesia language, a Thymelaeaceae, is widely found in Indonesia. Mahkota dewa has synonym of Phaleria papuana var warb wichnanmi (val) Back. In English, it is known as crown of God. Mahkota dewa fruit is frequently and empirically utilized by Indonesian to treat various diseases with satisfactory results. The bark and fruits are rich in saponins, alkaloids, polyphenolics, phenols, flavanoids, lignans and tannins. Isolated constituents of fruit include Phalerin, Icariside C3, magiferin, and gallic acid. From its seed, 29-norcurcubitacin and its derivatives, mahkoside A, mangiferin and kaempferol 3-O-β-D-glucoside had been isolated. In addition, phalerin was isolated from the methanol extract of its leave. Aglucon benzophenone and isophalerin were obtained from ethyl acetate extract of its leave. Ethyl acetate extract of its bark gave one compound of benzophenone glucoside.

The plant substances derived from the P. macrocarpa had been discovered to showcase high chemopreventive and antioxidant interest comparable to those of conventional drugs, and this activity has been attributed to the high phenolic content. P. macrocarpa is also found to have potent anti-inflammatory activity, due to its contents, including terpenoids, saponins, tannins, flavanoids and phenols such as rutin and cathecol.

This work reports the results of in silico molecular docking of Phaleria macrocarpa bark compounds (Fig.1) through inflammation pathway, because inflammation cause a signaling towards growth and proliferation cancer cell. To prove the docking result, we had extracted the bark of P. macrocarpa and further testing the cytotoxic activity against HCT116 cell line. The in vitro result then compared to in silico docking result.

Figure 1: Compounds of Bark Extract of Phaleria macrocarpa. Click here to view figure

Experimental

Preparation of Macromolecule

3D Structure of p50 NF-κB protein (PDB id: 1SVC), Interleukin-6 protein (PDB id: 1ALU), TNF-α (PDB id: 2AZ5), Interleukin-1β (PDB id: 1ITB), PPAR-α (PDB id: 1I7G), Interleukin-2 (PDB id : 1M48), MAPK (PDB id: 3LHJ), HIF-1 (PDB id: 4H6J) were downloaded from PDB (http://www.pdb.org/). Macromolecule was prepared to calculate the binding energy using Auto Dock tools. Water and non-standard residue were removed from the protein. Hydrogen and Gasteiger partial charges were further added to the carbon that held the hydrogen. The binding pocket of the protein was determined by grid based approach using default parameters. The grid map included the binding site along with the significant portions of the surrounding surface.

Preparation of Ligand

The structure of compound was made in 2D using Marvin Sketch 15.1.19 software and saved in 3D structure in. pdb format. The ligands then being optimized using Auto Dock tools to fix the charge, added the hydrogen and minimizing energy. The 3D structure then saved in. pdbqt format.

Molecular Docking and Their Interaction Studies

Molecular Docking of designed compounds was carried out using Lamarckian genetic algorithm in Auto dock 4.2 tools with default docking parameter. We selected Auto dock4.2 tool for the purpose of molecular docking because Auto Dock has established to be powerful tool able to quick and correctly predicting certain conformations and binding energies of ligands with macro molecular targets.

The success rate in retrieving binding modes of protein–ligand complexes is an essential validation for docking programs. The measure that is commonly used to determine whether or not a binding mode prediction was successful is the RMSD (8). RMSD between the lowest energy native ligand-docked pose and native ligand-native pose was evaluated using PyMol 1.7.4.5.

Docking interactions had been clustered to decide the Gibbs energy (ΔG) and optimum docking energy conformation was considered as the fine-docked pose. The generated conformations had related value of the Gibbs energy (ΔG). An estimated inhibition concentration (Ki) was used for determination of binding energies of various docking conformations, ranking according to their binding rankings (6).

Extraction and Fractionation Samples of Seaweeds

A total of 300 g of dry powder of Phaleria macrocarpa bark are macerated (soaked) with ethanol solvent in a sealed glass vessel for 3 days, with occasional stirring. Maceration is done three times to extract as much as possible of existing substances in sample of P.macrocarpa. The mixture was then filtered; the filtrate was concentrated using rotary evaporator. Furthermore, the extract were subjected to cytotoxic activity of the extracts was evaluated as a growth inhibitor of colorectal HCT-116 cancer cells by MTT assay.

Cytotoxicity Effects

The inhibition effect of synthesized compounds against colorectal cell line, HCT116, was tested using chemical 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide or MTT assay. First, in 96-well plates, cells were seeded at 5000 cells/well and incubated for 24 h. Media become renewed and the cells had been added with several concentrations of the extract (6–100 µg/ml) and Cisplatin as positive control (0–16 µg/ml)  incubated for a further 24 h. After 24 h, 20 µM of MTT solution (0.5 mg/ml MTT solution in media) had been added to each well and incubated for four hour at 37°C. The supernatant was aspirated and the MTT-formazan crystals formed by way of metabolically viable cells were dissolved in 100 µl of dimethyl sulphoxide (DMSO). In the end, the absorbance was monitored by a microplate reader at a wavelength of 570 nm. The percentage of viable cells was plotted versus the concentration of the test compound. The concentration by which to mediate 50% cytotoxicity (IC₅₀) was determined by linear regression analysis.

Result and Discussion

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