AIP Conference Proceedings 2193, 030003 (2019); https://doi.org/10.1063/1.5139340 2193, 030003
© 2019 Author(s).
Antiviral activity of Ceiba pentandra and Eugenia uniflora leaf extracts to dengue virus serotype-2 in Huh 7it-1 cell line
Cite as: AIP Conference Proceedings 2193, 030003 (2019); https://doi.org/10.1063/1.5139340 Published Online: 10 December 2019
Beti Ernawati Dewi, Marissa Angelina, Sabda Ardiantara, et al.
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Antiviral Activity of Ceiba pentandra and Eugenia uniflora Leaf Extracts to Dengue Virus Serotype-2 in Huh 7it-1 Cell
Line
Beti Ernawati Dewi
1,2,a), Marissa Angelina
3, Sabda Ardiantara
4, Agung Riyanto Prakoso
4, Hidayati Desti
1,2, and Tjahjani Mirawati Sudiro
1,21Department of Microbiology, Faculty of Medicine, Universitas Indonesia-Cipto Mangunkusumo Hospital, Jalan Pegangsaan Timur no 16, Jakarta, Indonesia.
2Infectious Disease and Immunology Cluster, Indonesian Medical Education and Research Institute, Faculty of Medicine, Universitas Indonesia, Jl Salemba no 6 Jakarta 10430, Indonesia
3Research Center for Chemistry, Indonesian Institute of Sciences. Kawasan Puspitek Serpong, Indonesia 15416
4Undergraduate student, Faculty of Medicine, Universitas Indonesia, Jl Salemba no 6 Jakarta 10430, Indonesia Corresponding author: a)[email protected]
Abstract. Dengue Fever (DF) is an infectious disease caused by dengue virus (DENV) infection which is transmitted by Aedes aegypti and Aedes albopictus mosquito bite. There are approximately 50 million DENV cases annually in the world with 500 thousand cases hospitalized. Incidence rate of DENV infection is increasing in Indonesia every year. Specific antiviral that can inhibit or mitigate the progression of the disease have been investigated for many decades. However, until now, management of DENV patients is only supportive. Specific antiviral drug to DENV is not available yet. The objective of this study is to determine antiviral activity of Ceiba pentandra and Eugenia uniflora leaf extract against DENV replication in Huh7it-1 cell line. Study design was an experimental study with a Focus assay and MTT assay to determine the effectivity and toxicity, respectively. Ceiba pentandra leaf extract showed IC50, CC50 and SI of 15.49 µg/ml, 81.1 µg/ml and 5.23. In other hand, E. uniflora revealed IC50, CC50, and SI of 19.83 µg/ml, 134.42 µg/ml and 6.78. We concluded that C. pentandra and E. uniflora leaf extract have potential as antiviral drug to DENV. Further studies are needed to explore the substance of C. pentandra and E. uniflora leaf extract that have antiviral activities to DENV.
Keywords: Ceiba petranda, Euginia uniflora, huh7it-1 cell line, denv-2
INTRODUCTION
Dengue hemorrhagic fever (DHF) is an infectious disease caused by the dengue virus (DENV) and is transmitted to humans by Aedes aegypti and Aedes albopictus mosquitoes. There are 2.5 million people have the risk of being infected with the DENV. An estimated 50 million cases of DENV infection occur annually in the world, with 0.5 million cases being hospitalized [1].Dengue virus infection is now endemic in more than 100 countries, especially in the Western Pacific region, America, and Southeast Asia including Indonesia [1]. In Indonesia, in 2015 there were 126,675 DHF cases and 1,229 of them died. The incidence rate of DENV continues to increase every year in Indonesia [2]. An estimated 500 000 people with severe dengue requires hospitalization and with an estimated 2.5% case fatality, annually.
Most of DENV infected patients are asymptomatic. DENV infection commonly showed a high fever (40°C/104°F) with accompanied by 2 of the following symptoms: severe headache, pain behind the eyes, muscle and joint pains, nausea, vomiting, swollen glands or rash. Severe DENV infection has a potentially deadly due to plasma leaking, fluid accumulation, respiratory distress, severe bleeding, or organ impairment. DENV infection in more severe cases can
cause DHF and dengue shock syndrome (DSS). Without any treatment, DSS can cause death [3]. For now, the management that can be given to patients with DENV infection is only supportive treatment of adequate fluid administration therapy. There is no specific antiviral therapy available for patients with DENV infection. The DENV has a life cycle that begins with the attachment of the virus to the host cell membrane, endocytosis, fusion, and RNA replication. The life cycle of the dengue virus can be used as a target for molecular treatment.
Indonesia has a variety of herbal medicine that have been used by community since ancient time. Herbal medicine contains numerous active constituents for therapeutic and have been used as an exemplary source for centuries as an alternative remedy for treating human diseases. Approximately 80% of the third world population depend on traditional medicines for maintaining general health and combating many diseases [2]
Herbal medicines that have been proven to control the DENV are Carapichea ipecacuanha, and Echinacea [4].
Other plants that are often found in Indonesia such as Carica papaya and Psidium guajava leaf have potential as antiviral to DENV [5]. In this study, we explored the potential of C. petandra and E. uniflora leaf extract as antiviral to DENV in vitro.
MATERIALS AND METHODS Preparation of Extracts
The botanical identity of C. pentandra and E. uniflora plant were identified and authenticated by botanist in Research Center for Biology, Indonesian Institute of Science (LIPI). After taxonomical identification of each plant, the leaf was washed and ground until turned into a fine powder according to previous study [6]. Stock was stored at - 20°C until further use.
Preparation of DENV2 and Huh7-it Cell Culture
Dengue Virus (DENV) serotype 2 strain NGC adapted in Huh7it-1 cell was used in this study. The propagation of DENV-2 according to previous study [6]. A monolayer of Huh7it-1 cell in T-75 flasks were infected with DENV-2 NGC at MOI of 0.5 PFU/cell and then the flask was incubated at 37°C with 5% CO2 for 5—7 days. The virus was filtered using a syringe driven 0.22 mm (Millipore Co. Bedford USA). Culture supernatant was stored at -80°C and checked for the titer of dengue virus by focus assay as previously performed by Igarashi et al, 1995 [7].
Determination of Half-Cytotoxic Concentration
In vitro cytotoxicity of each extract was determined by MTT assay that quantified the percentage viability of Huh- 7 cells after treated with a certain concentration of extract compared with negative control (DMSO) 0.1%). For cytotoxic assay, we used same method with previous study. We diluted C. pentandra and E. uniflora at various concentration of 160, 80, 40, 20, 10, and 5 µg/ml. The resulting percentage toxicity values of each concentration that was tested in triplicate was calculated for its mean and standard deviation and then the mean percentage was plotted to corresponding concentration to generate concentration-mean percentage of viability curve. A nonlinear regression equation was derived from the curve to calculate the half-cytotoxic concentration (CC50) of each leaf extract.
Determination of Half-Inhibitory Concentration
We used Focus Forming assay (FFA) to determine antiviral activity. The FFA is a variation of the plaque assay, but instead of relying on cell lysis to detect plaque formation, the FFA employs immunostaining techniques using labeled antibody specific for a DENV antigen to detect infected host cells and infectious virus particles before an actual plaque is formed. Therefore, in FFA, those DENV infected cells that commensurate with number of DENV, make foci with brown color after immunostaining. A total of 2×104 cells/well were seeded into 96-well plate and the plate were incubated at 37°C with 5% CO2. After 24 hours, the cells were infected with DENV-2 with MOI of 1 FFU/cell according to previous study [6]. In this study we used various concentration of extracts ranging from 5, 10, 20, 40, 80, and 160 µg/ml. Treatment with 0.1% of DMSO were used as negative control. Plates were further incubated at 37°C for 3 days. Next, viruses were harvested, and its titer were determined by Focus assay [6]. Briefly, the products of 10-fold serial dilution of the supernatant was inoculated onto Huh7 it-1 cell monolayer in triplicate wells.
Absorption was carried out at 37°C with 5% CO2 for 2 hours with agitation at 30 minutes interval. Methylcellulose 1.5% overlay medium was added to the cell and incubated at 37°C with 5% CO2 for 3 days. The infected cells were stained according to previous publication by Payne et. al. (2006) with slight modification [8]. First, infected cells were fixed with 10% formaldehyde in PBS and were incubated at room temperature for an hour. The cells were washed with PBS for three times. To make the cells permeable, a non-indent P40 1% were added 100 l/well and then the plate was incubated at room temperature for 30 minutes. Next, the cells were blocked with 5% skim milk in PBS and incubated at room temperature for an hour. After cell washing, human IgG-anti dengue were added to each well 1/1000 and incubated at room temperature for 1 hour. For the second antibody, 1/1000 antihuman IgG label HRP was used.
After washed using PBS, substrate for horseradish peroxidase were added and cells were observed for its brownish color. Number of foci formed in each well including in negative control well was counted manually under microscope after staining. Each concentration of extracts was tested in triplicate hence the mean value of percentage of infectivity for each concentration was calculated. Number of foci in each treatment well was compared to that of negative control well to obtain percentage of infectivity of each well. The mean value of percentage of infectivity for each concentration triplicate was calculated and then those values were plotted against corresponding concentration to generate concentration-percentage of inhibition curve. The half-inhibitory concentration (IC50) was obtained from nonlinear regression equation of concentration-effect curves.
Data Analysis
Mean difference of percentage of cytotoxicity and infectivity between treatments group and negative control was analyzed using One-way ANOVA using SPSS version 23 with p value less than 0.05 (p<0.05) considered as statistically significant difference. The value of CC50 and IC50 were determined using simple arithmetical calculation on regression equations obtained from concentration-percentage of viability and concentration-percentage of inhibition. Then, selectivity index for each extract was derived from the ratio of CC50 to IC50.
RESULTS
Cytotoxicity of C. pentandra and E. uniflora Leaf Extract on HuH7it-1 Cell Culture
Cell viability in this study was presented by the percentage of surviving cells after treated with extract compared to DMSO controls. Cell viability were measured by the MTT assay. The cell viability values were obtained by comparing the absorbance of the spectrophotometry of each test concentration with DMSO 0.1% absorbance at a wavelength of 490 nm, then multiplied by 100%. Cell viability values were used to determine half-cytotoxic concentration (CC50). The calculation results of cell viability were shown in Table 1. We found that addition C.
pentandra at concentration of 10 µg/mL and more reduced viability of cells. Similar result also found in addition of E. uniflora (Table 1). Addition of 80 µg/mL of C. pentandra, reduced viability of cell to 40.32% (Table 1). In other hand, the viability of cells went to 44.2% after addition of E. uniflora at concentration of 160 µg/ml.
TABLE 1. Mean percentage of Huh 7it-1 cell culture viability post-treatment with corresponding C. petandra and E. uniflora extract’s concentration
Concentration (μg/ml) Percentage of Huh7it-1 Viability C. pentandra E. uniflora
160 22,15 0,46 44.2 2.52
80 40,32 1,47 56.07 2.04
40 63,38 7,57 72.92 3.62
20 76,26 4,35 76.51 5.16
10 80,44 6,70 83.43 3.25
5 90,85 1,59 91.94 2.83
DMSO 0.1%* 100,67 6,40 100 1.90
*DMSO 0.1 % was used as the negative control as well as the denominator to which optical density of each well containing a certain amount of extract compared which generated percentage of viability of each well.
A nonlinear regression curve of each extract’s concentrations and cells viability was made and then the graph formula was made using Microsoft Excel 2013. Half-cytotoxic concentration (CC50) of C. pentandra and E. uniflora were determined from the equations obtained from respective graph. Half-cytotoxic concentration is defined as extract’s concentration that gives rise to the decrement of cell culture viability by 50% with respect to the negative control cells’ percentage of viability. The CC50 of C. pentandra and E. uniflora were 81.1 µg/ml and 134.424 µg/ml, respectively (Figure 1).
(a)
(b)
FIGURE 1. Concentration-mean percentage of Huh7it-1 cells viability regression curve post-treatment of (a) C. pentandra (b) E.
uniflora. From the regression equation obtained from corresponding curve, the CC50 of C. pentandra and E. uniflora were calculated
Infectivity of DENV-2 after Treated with C. pentandra and E. uniflora Leaf Extract on HuH7it-1 Cell Culture
The infectivity of DENV-2 was determined by Focus assay. The percentage of infectivity was presented by the number of DENV-2 after treated with extract compared to DMSO controls, then multiplied by 100%. We found that increasing of concentration of C. pentandra and E. uniflora leaf extract were reduced the infectivity DENV-2 (Table 2.). Addition C. pentandra at concentration of 80 ug/mL and more, the infectivity of DENV were 0%. Similar results were found in DENV-2 after treated with E. uniflora (Table 2.). The infectivity reduced when the concentration of extract was increased.
y = -0.4273x + 84.665 R² = 0.9347
0.00 20.00 40.00 60.00 80.00 100.00
0 50 100 150 200
Average of % Viability
Concentration (µg/mL)
y = -13.37ln(x) + 115.53 R² = 0.9586
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00
0 50 100 150 200
average of % Viability
Concentration (µg/mL)
TABLE 2. Mean percentage of DENV2 infectivity post-treatment with corresponding extract’s concentration Extract concentration (μg/ml) Percentage of DENV2 Infectivity
C. pentandra E. uniflora
160 0.0 0.0 1.37 0.47
80 0.0 0.0 1.92 0.47
40 1.0 0.8 4.11 0.82
20 15.0 6.0 28.49 5.02
10 64.7 12.3 41.37 25.27
5 107.7 20.9 133.97 72.20
DMSO 0,1%* 100 3.3 121.67 59.91
*DMSO 0.1% was used as the negative control to which number of foci formed on each well containing a certain amount of extract compared to obtain the value of inhibition percentage.
(a)
(b)
FIGURE 2. Regression curve for concentration-mean percentage of DENV2 post-treatment of (a) C. pentandra (b) E. uniflora.
From the regression equation obtained from corresponding curve, the IC50 of C. pentandra and E. uniflora were calculated. From the curve of the relationship between the percentage of infectivity and the concentration of C.
pentandra leaf extract, the equation y = -30.39ln (x) + 133.29 with the value of R² = 0.7774 was obtained (Figure 2a).
From the equation, we found that IC50 value, of C. pentandra was equal to 15.49 µg/ml. In other hand, the equation of E. uniflora was y = -33.21ln(x) + 146.21 with R² of 0.71. Therefore, the IC50 value of E. uniflora was equal to 19.826 µg/mL (Figure 2b).
y = -30.39ln(x) + 133.29 R² = 0.7774
-40.0 -20.0 0.0 20.0 40.0 60.0 80.0 100.0 120.0 140.0
0 50 100 150 200
Average % infectivity
concentration (µg/mL)
y = -33.21ln(x) + 146.21 R² = 0.7103
-50.00 0.00 50.00 100.00 150.00 200.00 250.00
0 50 100 150 200
average of % Infectivityi
concentration (µg/mL)
DISCUSSION
DENV infection still serious health problem in the world including Indonesia. Specific treatment is not available yet has hamper to reduce mortality rate. Various plants have been used by traditional healers and simultaneously been scientifically studies. Ceiba pentandra is known as silk cotton tree and normally found in the wild. Ceiba pentandra belongs to the family of Bombacaceae and traditional medicine [9]. Various parts of C. pentandra have been reported to be useful as effective against diabetes, hypertension, headache, dizziness, constipation, mental diseases, fever, peptic ulcer, and leprosy. The bark is believed to also contain tannin and have been used in the treatment of diarrhea [10,3].
Ceiba pentandra plants are found in Malaysia and Java. This plant has been used to cure many infectious diseases in traditional system of medicine. Many researchers had studied antimicrobial activity of other parts of C. pentandra such as bark, leaves and fruits that but still few study on antibacterial activities of seeds. Ceiba pentandra is a plant that can be used for antidiabetic treatment, antimicrobial agents, and treat wounds or abscesses, and has an antiviral effect. Ceiba pentandra leaf extract contains flavonoids and tannins which are antioxidant agents [7]. In addition, in a study it was found that increasing antiviral activity can occur with increased levels of flavonoids [8].
Beside C. petandra, E. uniflora also showed an antimicrobial, antioxidant function and antiviral activity due to the high content of flavonoids [10,11]. The presence of flavonoids will inhibit replication of DENV, which increases the concentration of flavonoid compounds with an increase in antiviral activity [12]. Ceiba pentandra the highest phytochemical compound was glycosides [18.71±2.0] present in the leaves and phenol[12]. Moreover, their proximate values were also established, with both plants having carbohydrate. In addition, the work tried to establish the mineral content of the plants, minerals like potassium, sodium, phosphorus, magnesium and calcium were tested for, although found in minute quantities [12].
The other herbal medicine such as Carica papaya alredy move as DENV treatment. C. papaya Leaf extract proven increase in the number of platelets due to the expression of arachidonate 12-lipooxygenase gene and platelet-activating factor receptor gene [13]. The low cytotoxic and high inhibition to DENV suggested that C. petandra and E. uniflora can be explored more as antiviral drug to DENV in future.
CONCLUSION
The leaf extract of C. pentandra and E. uniflora had shown potent antiviral activity to DENV2 in Huh7it-1 cell culture. The half-inhibitory concentration, half cytotoxicity concentration and selectivity index of C. pentandra was 15.49 µg/ml, 81.1 µg/ml and 5.24. The IC50, CC50 and SI of E. uniflora was 19.83 µg/ml, 134.42 µg/ml and 6.78 respectively.
Further studies are needed to be done to confirm and utilize the findings for the development of dengue antiviral agent. Among those required studies are in vitro study using the remainder serotypes of DENV, in vivo study, and study to explore the bioactive compounds as well as inferring its mechanism of action based on the treatment design of the research.
ACKNOWLEDGEMENT
This study was supported by grant of Publikasi Terindeks Internasional untuk Tugas Akhir Mahasiswa UI (PITTA) 2018 No: 0588/SK/R/UI/2018.
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