efek anti virus dari virgin coconut oil terhadap replikasi
TRANSCRIPT
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,
Efek anti virus dari Virgin ..., Shehla Mughal Endo, FK UI, 2014
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
Efek anti virus dari Virgin ..., Shehla Mughal Endo, FK UI, 2014
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).
Efek anti virus dari Virgin ..., Shehla Mughal Endo, FK UI, 2014
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)
Efek anti virus dari Virgin ..., Shehla Mughal Endo, FK UI, 2014
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.
Efek anti virus dari Virgin ..., Shehla Mughal Endo, FK UI, 2014
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
Efek anti virus dari Virgin ..., Shehla Mughal Endo, FK UI, 2014
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
Efek anti virus dari Virgin ..., Shehla Mughal Endo, FK UI, 2014
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
Efek anti virus dari Virgin ..., Shehla Mughal Endo, FK UI, 2014
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
Efek anti virus dari Virgin ..., Shehla Mughal Endo, FK UI, 2014
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
Efek anti virus dari Virgin ..., Shehla Mughal Endo, FK UI, 2014
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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