efek anti virus dari virgin coconut oil terhadap replikasi

Efek Anti Virus dari Virgin Coconut Oil terhadap Replikasi Virus Dengue

Shehla Mughal Endo1, Beti Ernawati Dewi, Ssi, PhD2

1. General Medicine, 2. Microbiology

ABSTRAK

Kasus Demam Berdarah Dengue (DBD) dan demam dengue (DD) dilaporkan meningkat di seluruh dunia setiap tahunnya, terutama di negara Asia Tenggara termasuk Indonesia Gambaran klinis dari DBD/DD adalah demam, sakit kepala, nyeri otot dan sendi, ruam kulit yang mirip dengan campak, dan hasil lab menunjukkan penurunan jumlah trombosit. Hingga saat ini belum ada antiviral khusus untuk DBD. Penelitian ini bertujuan untuk mengevaluasi pengaruh virgin coconut oil (VCO) terhadap replikasi virus dengue (DENV). Penelitian ini merupakan penelitian eksperimental yang dilakukan di Laboratorium Mikrobiologi, Departemen Mikrobiologi, Fakultas Kedokteran Universitas Indonesia. Data yang diperoleh ini berasal dari hasil eksperimen yang dilakukan dengan 6 pengulangan untuk setiap perlakuan yaitu pemberian VCO 5%, 1%, 0,5% dan 0,1%, kontrol negatif dan Dimethyl Sulfoxide (DMSO). Penghambatan replikasi DENV dilihat dengan menghitung titer virus setelah perlakuan VCO. Titer virus dihitung dengan menggunakan metode focus assay. Hasil penelitian menunjukkan bahwa IC50 dari VCO adalah kuat, sementara CC50 VCO adalah moderat. Hal ini menunjukkan bahwa secara signifikan VCO menghambat replikasi DENV dengan kisaran cukup aman untuk digunakan pada sel dalam dosis terbatas. Penelitian lebih lanjut perlu dilakukan untuk mengevaluasi efek VCO pada replikasi DENV in vivo, sehingga dapat ditemukan kandidat anti DENV di masa mendatang.

Keywords: Efek antiviral; CC50; IC50; Virgin coconut oil; Virus dengue; Cytotoxicity

Efek anti virus dari Virgin ..., Shehla Mughal Endo, FK UI, 2014

The antiviral effect of vigin coconut oil to the replication of dengue virus in vitro

Shehla Mughal Endo1, Beti Ernawati Dewi, Ssi, PhD2

1. General Medicine, 2. Microbiology

ABSTRACT

Cases of the Dengue Hemorrhagic Fever (DHF)/Dengue Fever (DF) were reported increasing worldwide annually, especially in South Asia counties including Indonesia The clinical features of DF/DHF are fever, headache, muscle and joint pains, a characteristic skin rash that is similar to measles, which lead to thrombocytopenia as a lab result. Until now, specific antiviral for dengue virus (DENV) is not available yet. The objective of this research was to evaluate the effect of virgin coconut oil (VCO) to the DENV replication. This research was experimental study and was conducted at Microbiology laboratory, Department of Microbiology, Faculty of Medicine University of Indonesia. The data that was obtained for this study came from the experimental studied with 6 repeated experiments for each treatment of various concentartion of 5%, 1%, 0.5% and 0.1% as well as negative control and Dimethyl Sulfoxide (DMSO). Inhibition of DENV replication was determined by calculating of DENV titer after treated with VCO. The focus assay was used to calculate the DENV titer. The result showed that IC50 and CC50 of VCO was strong and moderate respectively. VCO was significantly inhibited the replication of DENV with adequate safe range to use for cells within limited dosages. Therefore, we concluded that VCO can be a candidate antiviral for DENV. Next study is needed to evaluate the effect of VCO in vivo, therefore we will find an antiviral of DENV virus in future.

Keywords: Antiviral effect; Cytotoxicity; Dengue virus; CC50; IC50;

Virgin Coconut oil,


INTRODUCTION

Background

The DENV infection have found in tropical and subtropical regions

predominantly in urban and semi-urban areas.1 (1) The disease is caused by infection of

DENV belonging to the family of Flaviviradae and spreading by Aedes mosquitoes.

There are approximately 500 000 people with DHF requiring hospitalization out of 390

million estimated DENV infection annually.2(2)

Even though its high incidence, challenges to find out antiviral against DENV

virus have not been succeed yet until today because there is limited understanding of

how the pathogens typically behaves and how the virus interacts with the immune

system. Since there is no reliable vaccines or therapeutics for DENV, development of

effective way to manage DENV remains high global public health priority.

From those reasons, the research of antiviral against DENV is highly desired.

Several natural products are currently considered as antiviral to replication of DENV

such as virgin coconut oil. Interestingly, several recent researchers have described its

antiviral effect against several viral replications.9(9)In this research, I found the antiviral

effect of virgin coconut oil to replication of DENV. The development of coconut oil as

antiviral to dengue viruses may promise a more potential alternative in combating

dengue hemorrhagic fever.

3


LITERATURE REVIEW

DENV

DENV is members of Flaviviridae family, including some viruses that pose a threat

to public health, including yellow fever virus, West Nile virus, Japanese encephalitis

virus and tick-borne encephalitis virus.12 (12)

Each of the four serotypes of DENV (DENV-1, DENV-2, DENV-3 and DENV-4) is

capable to produce the full spectrum of clinical manifestations following DENV

infection.13 All of them possess single-strand RNA genome. Infection with any one

serotype confers lifelong immunity to the viral serotype.12 Although all four serotypes

are genetically similar, they are different enough to elicit cross-protection for only a few

months after infection by any one of them.3 The secondary infection caused by another

serotype or multiple infections with different serotypes predispose to severe form of

DENV.3 (3, 13).

DENV infection

DENV is considered as a febrile illness that may bring fatal outcome in severe cases.

The DENV appears in two forms; the first classic DENV with mild to high fever,

retro-orbital pain, severe headaches, maculopapular rashes, muscle and joint pain.

Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS), are severe

forms of DENV infection, which is typified by high fever, thrombocytopenia (reduced

thrombocyte), abdominal bleeding, hemorrhage and circulatory failure, which is fatal

without proper management.13,15 (13, 15).


Antiviral effect of Coconut oil

As scientific field develops, several novel effects of coconuts have been reported

such as anti-inflammatory, anti-hypertensive or even anti-osteoporosis effect.7,28-30 The

most important and interesting effect is antiviral effect.31,32 Several research have

proved that coconut oil is very effective against a variety of viruses especially which

have lipid-coated such as visna virus, CMV, Epstein-barr virus, influenza virus,

leukemia virus, pneumono virus as well as hepatitis C virus.32 The medium-chained

fatty acids (MCFA) in coconut oil primarily destroy these viruses by disrupting their

membranes, interfering its assembly and maturation. There are two leading substances,

which confer antiviral effect of coconuts oil; lauric acids and monolaurin.32 Lauric acid

which are saturated fatty acids and comprises about half of fatty acids content in

coconuts oil has greater antiviral activity. Similar to lauric acids, the monoglycerides are

active while diglycerides and triglycerides are inactive against these viruses.

Monolaurin which is a main monoglyceride in the coconuts oil acts by solubilizing the

lipids and phospholipids in the envelope of the virus, causing the disintegration of the

virus envelope.31 Inconsequence of these functions, coconuts oil is critical for

inactivating viruses.(28-32)


METHOD

Method

We used Vero cell (money kidney cell line) because it was very sensitive to DENV. We

prepared 6 groups with 6 repeated of Vero cells in 96 well plate including DMSO, and

negative control. Each Vero cell group was prepared as monolayer to prevent hypoxic

cell death due to multilayer and made easier for DENV to attach and infect the cell.

Determination of IC50

The IC50 was obtained from nonlinear regression analysis of concentration-effect curves by the graph and represented the means ± standard deviation experiments. Determination of CC50 The absorbance reading was measured using the microplate reader at 490 nm. Hence, the result of percentage of the cell viability and toxicity was determined by the absorbance reading. Data Analysis

Statistically analysis of data was done using T-test to determined difference in each

group, compared to DMSO. We used both Microsoft Excel and SPSS to confirm

calculated t-value If the calculated p-value was less than 0.05, difference between

control and treated groups were significant.


RESULTS

4.1. Foci of DENV

Figure 1 Focus assay A: 5% VCO treatment, B: 1% VCO treatment, C: 0.5% VCO treatment, D: 0.1% VCO treatment, E: DMSO, F: Negative control

From focus assay, we found DENV infected cells (foci) with the brown colour. To

stain the dengue virus, immunoperoxidase staining was used for this research since it

provides morphological details and immunological identification. It was able to stain

the virus because antibodies against DENV. Addition of Methycellulose in focus assay

to infected vero cell gave result that one foci come from one DENV.

The negative control did not show any focus in its wells refeal that negative control in

this experiment was not contaminated with DENV. As Figure 1 shows that focus assays

of each 1-0.1% concentration of VCO treatment and DMSO were not obvious

difference between them. It suggests that the significant inhibitory effect of VCO whose

concentration range from 1- 0.1% was not found. While comparing the 5% VCO, we

found decrease of foci number.

4.2. Titer of DENV after VCO treatment

The result of cell culture with dengue virus, which was treated with virgin coconut oil is

listed in Table 4.1.

D E A B B


Table 1.1 Average Titer and SD of different VCO concentration, DMSO and negative control

VCO treatment Average of Titer (FFU/mL) ± SD

VCO 5.0% 9.67×104 ±6.95×104

VCO 1.0% 10.09×104 ±2.48×104

VCO 0.5% 1.39×104 ±2.66×104

VCO 0.1% 1.33×104 ±1.29×104

DMSO 2.79×104 ±1.9×104

Negative Control 0

4.3 Inhibition of DENV by using VCO

Table 4.2 2Average Inhibition (%) and SD

VCO treatment % Inhibition average ± SD

VCO 5.0% 65.4 ±24.89

VCO 1.0% 63.8 ±8.89

VCO 0.5% 50.2 ±9.53

VCO 0.1% 52.2 ±4.61


By using Microsoft Excel, the following equation was obtained.

Figure 2 Equation of IC50

As illustrated in Figure 2, if y-axis is applied for 50, IC50 was determined as -0.1259.

The criteria of strong, moderate and low inhibition were less than 2ug, between 10ug &

2ug as well as more than 10ug, respectively. Since IC50 of VCO was able less than

0.1ug, it was categorize as strong, which means that it showed significant inhibition

against DENV replication. Therefore, it is able to conclude that inhibitory ability of

VCO was high.

4.4 Cytotoxicity of VCOs

The Table 4.3 indicates the result of treatment by using VCO. By using the formula

which requires values of OD (optical density), the following result was obtained.

y = 2.9779x + 50.375 R² = 0.96247

0 10 20 30 40 50 60 70

0 2 4 6


Table 4.3 3 Average of Optical Density, Viability (%) and Standard deviation

VCO treatment OD Average Viability% ± SD

VCO 5.0% 1.13 104.73±0.08

VCO 1.0% 1.16 107.90±0.06

VCO 0.5% 1.23 116.19±0.1

VCO 0.1% 1.16 107.85±0.05

DMSO 1.07 100±0.12

By using the Microsoft Excel, following equation was obtained.

Figure 3 Equation of CC50

In respect to figure 3, when 50 was applied for y-axis, CC50 was determined as 33.98.

The CC50 criteria of toxic, moderate and less cytotoxic are less than 10ug, between 10ug

& 50ug as well as more than 50ug, respectively. Therefore, CC50 of VCO is considered

as moderate.

y = -1.8738x + 113.67 R² = 0.60999

100

105

110

115

120

0 2 4 6


DISCUSSION

Nowadays, there are many researches which identified the antiviral properties of virgin

coconuts oil due to its abundant medium chain fatty acids (MCFAs), including capric

acid, caprylic acid, as well as the powerful lauric acid, which attack and kill viruses with

lipid coatings. These fatty acids are concentrated in coconut oil so that it comprises

approximately 60 percent of all components in the oil. The VCO shows moderate to

strong antiviral properties to lipid-coated virus such as herpes, measles, HIV, hepatitis

C, influenza, and mononucleosis because lauric acid and capric acid are metabolized

into the monoglycerides monolaurin and monocaprin, which is small enough to be

considered useful by the viruses’ coating, but too large to actually be of any benefit.

Then, they attach to and then dissolve this protective coating, essentially spilling their

insides out and killing the viruses. This works against many harmful bacteria and fungi

as well while being harmless to human cells. Although VCO have antiviral effect to

certain lipid-coated virus, it may not cure many illnesses, but it seems to lessen the viral

load many infected have to carry since, generally speaking, the antiviral effect of VCO

is not significant enough in order to treat and diminish the clinical features of infection.

VCO did exhibit the strong antiviral effect against DENV because the surface pore of

DENV was large enough to pass the monoglycerides monolaurin and monocaprin,

which are the metabolites of lauric acid as well as capric acid. Therefore, VCO was able

to enter the interior of DENV so that it could induce the strong anti-replication of

DENV. As a result of our experiment, CC50 of VCO was able to categorize as moderate,

which meant that it did not show strong cytotoxicity against cells as long as the dose of

VCO was not extremely high. It was obvious that the most suitable treatment for human


was better to utilize the less cytotoxic reagent (CC50 is less than 2ug). However,

moderate agents were also able to use due to its relative safeness. Therefore, it is still

safe for VCO to use as a clinical treatment since it exhibited moderate CC50 and strong

IC50 against DENV.

CONCLUSION AND RECOMMENDATION 5.1 Conclusion

The result of this experiment revealed that the antiviral effect of virgin coconut oil

(VCO) in order to suppress the growth of DENV in vitro was very strong since its

IC50 was less than 0.1ug. Furthermore, the CC50 of VCO was moderate. Taken

together, possibility to use VCO as a clinical treatment of DENV infection is highly

recommend in consequence of relative safety and strong inhibitory effect of VCO.

Therefore, my research hypothesis “Virgin coconut oil has antiviral effect to DENV

in vitro” was accepted.

5.2 Recommendation

As a recommendation, I propose to perform in vitro research with different

concentration of VCO to clarify the effect of VCO in vitro against DENV infection.

Although it is still required to confirm VCO function in vitro, clinical usage of VCO

as a treatment of DENV is highly possible owing to its strong anti-replication effect

and relative safety to use, as shown in this research.

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