AIP Conference Proceedings 2193, 030004 (2019); https://doi.org/10.1063/1.5139341 2193, 030004
© 2019 Author(s).
In vitro and in silico study to evaluate the effectiveness of quercitrin as antiviral drug to dengue virus
Cite as: AIP Conference Proceedings 2193, 030004 (2019); https://doi.org/10.1063/1.5139341 Published Online: 10 December 2019
Beti Ernawati Dewi, Edianti Ratningpoeti, Hidayati Desti, et al.
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In vitro and in Silico Study to Evaluate the Effectiveness of Quercitrin as Antiviral Drug to Dengue Virus
Beti Ernawati Dewi
1,2,a), Edianti Ratningpoeti
3, Hidayati Desti
1,2, Marissa Angelina
41Department of Microbiology, Medical Faculty Universitas Indonesia-Cipto Mangunkusumo Hospital Jalan Pegangsaan Timur no 16, Jakarta, Indonesia.
2Infectious Disease and Immunology Cluster, Indonesian Medical Education and research Institute, Jl Salemba no 6 Jakarta 10430, Indonesia
3Undergraduate student, Medical Faculty Universitas Indonesia Jl Salemba no 6 Jakarta 10430, Indonesia
4Research Center for Chemistry, Indonesian Institute of Sciences, Kawasan Puspitek Serpong, Indonesia 15416
a)Corresponding author: [email protected]
Abstract. Dengue Fever (DF) in Indonesia has been a major health problem for the last 47 years, which keeps on rising until today. Quercitrin as one of the natural compounds, flavonol group and has been used in several types of research for their properties of anti-inflammation, anti-bacterial, anti-viral, and anti-fungi. The aim of this study was to explore the potency of quercitrin as an antiviral drug to dengue virus (DENV) in vitro and in silico. We used Focus assay, MTT assay, and docking analysis to determine IC50, CC50 and binding energy, respectively. The IC50, CC50 and Selectivity Index (SI) of quercitrin was 1.1 g/ml, 38.8 g/ml and 38 respectively. In silico study showed the binding energy between quercitrin and NS5 protein was -7,54 kkal/mol. The results obtained that Quercutrin as a good candidate for an antiviral drug to DENV in the future.
Keywords: DENV-2, in silico, huh7 it-1 cell line, quercetrin.
INTRODUCTION
Tropical countries tend to have a higher humidity level than non-tropical countries. Humidity is one of several factors that make the place suitable for mosquitos breeding. One of the most concerning the illness of mosquito-borne disease in the world is dengue hemorrhagic fever (DHF), which caused by dengue virus (DENV). There were up to 390 million cases per year with 500.000 infected people need to be hospitalized [1]. In Indonesia, the cases began to be reported from 1968 with 58 cases with case fatality rate (CFR) of 41.37%. In 2015, the CFR was 0,067% from 126.675 cases reported [2, 3].
Unfortunately, up to now, there is still no commercially antiviral drug available. The development of antiviral drug should effective to inhibit DENV replication as well as safe to consume them. The antiviral activities include blocking the viral entry or virus attachment to human cells [4].Theoretically, all proteins involved in the DENV replication cycle can be used as a potential antiviral therapy target. DENV consists of structural (C, preM, E) and nonstructural protein. (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) [5,6]. Recently, research has been conducted on NS5 protein, the biggest NS protein in flavivirus, as an important target. NS5 has 2 domains, RNA methyltransferase (MTase) on the end of N-terminal and RNA-dependent RNA polymerase domain (RdRp) on the end of C-terminal. MTase and RdRp play a role in viral RNA capping and a vital role in RNA replication. As there is no activity found of RdRp on the host cell, NS5 is believed as a specific antiviral target with a low level of toxicity [5].
One of many pure compounds that can be found in nature is quercitrin, even though the potency as an antiviral drug to DENV still remains unknown. Quercitrin is a flavonoid glycoside can be found mostly in plants and fruits such as apple, berries, grapes, onions, tomato to black tea. Studies have shown that flavonoid-O-glycosides are converted into the aglycone by bowel flora [6]. It has been used as anti-inflammation, anti-bacterial, anti-viral, and anti-fungi. As for its anti-inflammation properties, in vivo effects of quercitrin in the experimental model of rat colitis induced by dextran sulfate sodium can be mediated by the release of quercetin generated after glycoside's cleavage by the intestinal microbiota [7]. Quercitrin plays a positive role in carbohydrate metabolism and antioxidant status in diabetic rats [8]. The potency of Quercitrin as an antiviral drug to DENV need to be conducted. In this study, we will explore the potency of quercitrin in vitro using DENV serotype 2 strain New Guinea C (DENV-2 NGC) and human cell line Huh 7it-1. In addition, we also evaluated the potency of quercitrin in silico with NS-5 of DENV as a target.
MATERIALS AND METHODS Preparation of Quercitrin
We used commercial pure compound of Quercitrin and diluted in dimethyl sulfoxide (DMSO) at a concentration of 100 mg/mL as a stock solution. Further serial dilutions were made in DMEM with 2% FBS (infection medium).
Propagation of Cell Line and DENV
The research is conducted in Virology and Molecular Biology Laboratory, Department of Microbiology, Faculty of Medicine Universitas Indonesia. Huh, 7it-1 cells were cultured in T-75 Flask and incubated in 37°C with 5% CO2. Suspension cells in DMEM 10% FBS medium at a concentration of 4 x 104 cell/mL were added in 48 well plates for inhibitory evaluation. For toxicity evaluation, we used 96 well plate with the final cell number of 2 x 104 cell/well.
DENV serotype 2 strain new guinea C (DENV-2 NGC) were used in this study [9].
Determination of Antiviral Activity
Antiviral assay in this research was determined by focus forming assay (FFA) [9]. The antiviral activity of Quercitrin was done in monolayer cell in 48 well plates according to the previous study [10]. The cells were infected with DENV at MOI of 1 FFU/cell and added with various concentration of Quercitrin ranging from 10, 20, 40, 80, 160 until 320 µg/mL, for two hours. Afterward, added with a mixture of 200 µl/well of DMEM and 2% FBS along with a various concentration of Quercitrin, in triplicate. The plates were further incubated for 2 days at 37°C with 5%
CO2. The supernatant was harvested and the titer virus was determined by focus assay as the previous method [10].
Infectivity percentage of each Quercitrin concentration will be calculated by comparing the number of foci in each well to the negative control. The calculated mean value of infectivity percentage will be plotted to corresponding concentration to from inhibition curve. The regression of the non-linear equation of concentration-effect curves will be utilized to obtain the half-inhibitory concentration of the IC50[10].
Determination of Cytotoxicity
The toxicity level of Quercitrin to the cell was determined by MTT assay that evaluate the percentage of Huh 7it- 1 cells viability after being treated with Quercitrin and DMSO 0,1% as the negative control. The monolayer cells in the 96 well plate ware treated with various concentration of Quercitrin which ranging from 10, 20, 40, 80, 160 until 320 µl/mL and will be incubated again in 37°C temperature with 5% CO2 for 48 hours. MTT assay was done using the previous method [10]. The result of toxicity percentage of each concentration will be calculated to evaluate the mean value and standard deviation value, then the mean of the percentage will be plotted to corresponding concentration to form a concentration-mean percentage of viability curve. From the curve, the intersection of the concentration of Quercitrin and the regression of cell viability will determine the half-toxicity concentration (CC50) of Quercitrin.
Molecular Docking Study
We used the Autodock 4.2 program for docking studies with NS-5 protein as a target. Molecule 3D of NS-5 protein obtained from Protein Data Bank (http://www.rcsb.org) (PDB entry 1L9K4). Macromolecules that have been superposed were prepared for tethering. Macromolecules were optimized with software Autodock 4.2. 3D structure of macromolecules was added with hydrogen atoms. Then, their charges were repaired by adding the partial charges of Gasteiger Charges. Finally, they were given force-field of Autodock [11].
First, we optimized the macromolecular and ligand structure. After that, PDBQT files were created for both macromolecule and ligand, followed by grid parameter file (GPF) and a docking parameter file (DPF). GPF would inform Autogrid about potential receptors that need to be calculated and the type of map that must be counted and its location. A grid box of the protein structure was then generated using auto grid 4 software with (X, Y, Z), volume grid 50 x 50 x 50 Å, grid spacing of 0.375 Å. While the DPF would inform Autodock about the map that would be used, the ligand to be moved, including the center and the torque of the ligand, docking algorithm to be used, and the number of docking to be done. The binding mode was the main factor in the prediction of the binding affinity between quercitrin and IL9K4. This can be seen from the relatively highest ΔG and Ki values [12].
The binding free energies (ΔGbind) were estimated via the widely used Molecular Mechanic/Poisson Boltzmann Surface Area (MM/PBSA) method. According to this approach, the free energy was calculated for each ligand/protein system as described by the following equations:
∆𝐺𝑏𝑖𝑛𝑑 = 𝐺𝑐𝑜𝑚𝑝𝑙𝑒𝑥 − (𝐺𝑝𝑟𝑜𝑡𝑒𝑖𝑛 + 𝐺𝑙𝑖𝑔𝑎𝑛𝑑) = ∆𝐻𝑏𝑖𝑛𝑑 − 𝑇∆𝑆𝑏𝑖𝑛𝑑 (1)
The entropic contribution was obtained by performing normal mode analysis in order to be able to calculate the total ΔG [12].
RESULT
IC
50value of Quercitrin
The effectivity of quercitrin as an antiviral drug to DENV was determined by Focus assay. The focus assay of each concentration of extracts was tested in triplicate and was calculated compared with treated DMSO as a negative control. From Focus assay, we found that titer DENV after treated quercitrin in dose-dependent manner. Decreased concentration of quercitrin showed increasing the infectivity. At concentration of 320 mg/mL the infectivity of DENV was 1.7% ± 0.3 (Table 1).
TABLE 1. The percentage of DENV infectivity after treated with various concentration of Quercitrin.
Concentration
(ug/mL) Average of Infectivity
(%) SD
320 1.7 0.3
160 25.5 0.7
80 31.4 0.4
40 39.1 0.8
20 48.4 1.6
10 56.3 2.6
To determine IC50 value, a regression curve of extract concentration-DENV2 inhibition percentage was made using Microsoft Excel 2013 and the formula for the graph was generated (Figure 1.). The half-inhibitory concentration of each extract was calculated from its respective formula. From the regression equation obtained from the corresponding curve, the IC50 of quercitrin was 1,1 μg/ml.
FIGURE 1. Regression curve for concentration-mean percentage of DENV2 post-treatment of quercitrin. The equation of this regression was y= 14.394ln(x) + 8.1814 with R² = 0.94138
CC
50value of Quercitrin
To determine the Cytotoxicity of quercitrin on Huh7it-1 cells, we were measured using MTT assay. The mean percentage of Huh7it-1 viability for each concentration in triplicate were calculated in comparison with the percentage viability cell treated with DMSO. Treated with Quercitrin at a concentration of less than 160 ug/mL did not show toxic to the Huh 7it-1 (Table 2.). The toxicity effect showed in dose-dependent manner.
TABLE 2. Average of percentage of viability cells after treated with various concentration of quercitrin.
Concentration (µg/ml)
Percentage
of viability (%) SD
10 163,431 10,2
20 133,1502 12,3
40 141,2088 13,4
80 109,707 8,7
160 107,0208 6,7
320 42,97924 7,6
FIGURE 2. Average of percentage of viability cells after treated with various concentration of quercitrin were regression curve post-treatment.
0 50 100 150 200
0 100 200 300 400
P er cen tag e o f vi abi lty (%)
Concentration (µg/ml)
A nonlinear regression curve of each concentration and cells viability was made and then the graph formula was made using Microsoft Excel 2013. Half-cytotoxic concentration (CC50) of quercitrin was determined from the equations obtained from the respective graph (Figure 2). From the regression equation obtained from corresponding curve (y= -0.2684 + 144,78 and R2= 0,8051), the CC50 of quercitrin was 38.8 μg/ml. Based on IC50 and CC50 value we found that SI of quercitrin was 38
In Silico Study
To predict the potency of Quercitrin as an antiviral drug to DENV, we conduct in silico study to NS-5 protein of DENV. The binding interactions quercitrin into the active site of NS protein (IL9K4) modelled protein resulted in energy conformation of -7.67 kkal/mol. The residues Lys181, Ile147, Gly 148, Gly 181, Phe133, Val132, Thr104, Lys105 were important for strong hydrogen bonding interaction with quercitrin
FIGURE 3. Ligand interaction quercitrin with amino acid residue of NS5 protein (IL9K4).
DISCUSSION
Research in the development of the antiviral drug to DENV still challenging since there was no specific antiviral drug to treat DENV infection yet. Quercitrin is a plant-derived polyphenolic flavonoid glycosides which found in many fruits and vegetables, able to improve mental and physical performance and reduce infection risk due to biological properties. Biological properties have many benefits for overall health and diseases resistance including anti-carcinogenic, anti-inflammatory, antiviral, antioxidant and psychostimulant activities. Other than that, Quercitrin also able to inhibit lipid peroxidation, platelet aggregation and capillary permeability, also stimulate mitochondrial biogenesis.
Quercitrin also widely distributed in many varieties of foods such as apples, berries, Brassica vegetables, capers, grapes, onions, shallots, tea, tomatoes, nuts, flowers, barks and leaves. Several types of research that have been conducted shown that Quercitrin able to inhibit the production of lipopolysaccharide (LPS)-induce tumor necrosis factor α (TNF-α) in macrophages and downregulation of vesicular cell adhesion molecule 1 (VCAM-1) and CD80 expression [9,10].
Although the Quercitrin antiviral mechanism remains unknown, from this study proposed that Quercitrin inhibit the dengue virus replication. As the other flavonoids have effect against RNA polymerase and formation of complex with RNA, researchers suggested that Quercitrin may perform similar mechanism in disrupting viral replicationOther studies conducted by Saptawati et al. (2016), also suggested that extract C. papaya which impregnated by Quercitrin can inhibit DENV-2 replication by inhibition of LLC-MK2 and inhibition of NS2B/NS3 protease. Moreover, the study has revealed that Quercitrin has no cytotoxic effect [11].
Quercitrin able to perform antiviral properties in other viruses, such as in influenza virus. For influenza virus, Quercitrin reduced mRNA transcription in a dose-dependent manner. Quercitrin was also able to inhibit early stage of viral infection in the influenza virus, as it is shown to inhibit viral envelope protein hemagglutinin (HA) expression.
and inhibit HA2 subunit which aids the membrane fusion process during virus entry through inhibit influenza-virus- HA-mediated hemolysis.[12]
In rhinovirus, researchers also revealed that Quercitrin performs inhibition of viral replication at various stages, from blocking endocytosis via PI-3 kinase inhibition. Moreover, Quercitrin inhibits negative-strand RNA production through inhibition of RNA polymerase 3DPOL. Other research conducted also revealed that Quercitrin was able to
cleave eIFG4 to inhibit viral genome translation and inhibit PI-4-kinase which required for viral replication.
Furthermore, Quercitrin also caused a reduction of rhinovirus-induced pro-inflammatory cytokines such as IL-8 and IFN in airway epithelial cells, as well as airway cholinergic hyperresponsiveness [13,14]. Quercitrin decreased viral genome and hepatitis C virus infectious particles [15].
Description binding affinities of the NS-5 protein and quercitrin can be seen in figure 3. The binding interactions quercitrin into the active site of NS-5 modeled protein resulted in energy conformation of −7.67 kkal/mol. We suggested that quercitrin has a strong binding with NS-5 protein and could be as one of an inhibitor of DENV through binding with NS-5 protein. NS5 of DENV consists of RNA-dependent RNA polymerase (RdRp) and methyltransferase (MTase) and domains, which RNA synthesis and catalyze 5’-RNA capping/methylation, respectively, during replication. In silico study, molecular modeling to determine the specific interaction between macromolecule/protein and inhibitors. This in silico study was performed because there are difficulties on experiment directly on virus/host. Docking simulation not only presented an understanding of the binding mode of the ligands but it was also employed to validate the homology model [16]. To complete this study, in vivo study will be required to confirm in silico study. In vitro study, the half-inhibitory concentration, half cytotoxicity concentration and selectivity index of quercitrin were 1,1 µg/mL, 351,8 µg/ml and 319,8 respectively. This results showed that quercitrin has potential as an antiviral drug to DENV. Further studies are needed to explore the DENV inhibition mechanism by Quercitrin.
CONCLUSION
The IC50, CC50, SI and energy conformation of quercitrin was 1.1 µg/mL, 38.8 µg/ml, 38 and -7.67 kkal/mol respectively. We concluded that quercitrin had potential as an antiviral drug and can be a promising candidate of an antiviral drug to DENV in the future.
ACKNOWLEDGEMENT
This study was supported by grant of Publikasi Terindeks Internasional untuk Tugas Akhir Mahasiswa Universitas Indonesia (PITTA) 2019 No: 0588/SK/R/UI/2019.
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