In Vitro Study of Eight Indonesian Natural Extracts as Antiviral Against Dengue Virus

Background: Dengue hemorrhagic fever (DHF) caused by a dengue viruses is still a major problem in tropical countries, including Indonesia. World Health Organization data showed that over 40% of world population are at risk of DHF.1In 2014 there were 71.668 of DHF cases in 34 provinces with 641 death.2 In Central Java in 2013, the incidence rate and fatality rate of DHF was 45.52 in 100.000 populations and 1.21% respectively.3 Until nowadays, there is no vaccine or effective therapy is available as yet.4 Thus research on discovering specific antiviral against dengue is needed. Indonesia is rich in indigenous herbal plants, which may has potential antiviral activity, such as Psidium guajava (Jambu biji), Euphorbia hirta (Patikn kerbau), Piper bettle L (Sirih), Carica papaya (Pepaya), Curcuma longa L(Kunyit/turmeric), Phyllanthus niruri L (meniran), Andrographis paniculata (Sambiloto), Cymbopogon citrates (Serai). Previous studies show that these plants have antiviral and antibacterial properties.5However, there is only limited study of these plants against dengue virus . Objective: This study aimed to know whether these plants have potential activity against dengue virus in vitro. Method: Leave extracts of eight indigenous herbal plants as mention before were originated from Solo, Central Java, the crude extracts were tested in vitro against dengue virus serotype 2 (DENV-2) strain NGC using Huh7it-1 cell line. Those crude extracts were screened for antiviral activity using doses of 20mg/ml. Candidates that showed inhibition activity were further tested in various doses to determine IC50 and CC50. Result: From eight leave extracts tested, one of them i.e Carica papaya (pepaya) inhibited virus replication up to 89,5%. Dose dependent assay with C.papaya resulted in IC50, CC50 and selectivity index 6,57 μg/mL, 244,76 μg/mL and 37, 25 μg/mL respectively. Conclusion: C.papaya has potential antiviral activity against dengue virus in vitro. Further study is needed to confirm antiviral activity in vivo.

Dengue hemorrhagic fever (DHF) is an infectious disease which is still a major problem in tropical countries, including Indonesia. It is caused by dengue viruses that belong to Flaviviridae. WHO showed that over 40% of world populations are at risk of DHF. 1 In 2012 number of cases were 57,204 with 1229 death in South East Asia. 2 Indonesian Ministry of Health data showed that the number of patients with DHF increased annually. 3 In 2014 there were 71668 of DHF cases in 34 provinces with 641 death. 4 In Central Java in 2013 incidence rate and fatality rate of DHF was 45.52 in 100.000 populations and 1.21% respectively, which was increasing from the previous year 19.29 in 100.000 population and 0,93%. 5 Dengue appears in two forms, classic dengue fever and severe form. In severe form, Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS) which may cause abdominal bleeding, hemorrhage and circulatory failure. If this severe form is not treated with prompt and proper management it will lead to the fatal cases. 6 In addition, the morbidity and mortality from DHF is remaining high since there is no specific therapy for dengue. The management of cases is mainly supportive by fluid therapy. Thus, research on discovering specific antiviral against dengue is needed. Indonesia is rich in indigenous herbal plants, which may has potential antiviral activity, such as Psidium guajava (Jambu biji), Euphorbia hirta (Patikan kerbau), Piper bettle L. (Sirih), Carica papaya (Pepaya), Curcuma longa L. (Kunyit/ turmeric), Phyllanthus niruri L. (meniran), Andrographis paniculata (Sambiloto), Cymbopogon citratus (Serai).
Psidium guajava (Jambu biji) contains a number of major active compounds which play a role for major biological activities, including anti-inflamatory, antiplasmodial and antimicrobial properties. 7 Other study by Gyuris et al. (2009) reported that Euphorbia hirta has antiviral activities. The aqueous and 50% MeOH extracts of this plant inhibit replication of SIV mac251 (simian immunodeficiency virus strain mac251), HIV-1 and HIV-2 viruses (human immunodeficiency virus types 1 and 2) in MT4 human T-Lymphocyte cells lines. 8 Review on Piper bettle L. by Rekha et al. (2014) reported that the leaves of this plant have a tremendous potential biological activities such as antibacterial, antilarvasidal, immunomodulatory activities, etc. 9 Sarala and Paknikar (2014) on their scientific literature review suggested that Carica papaya leaf extract have beneficial properties in dengue due to flavonoids and other phenols compound. The Carica papaya leaf extract also rich several minerals which may balance the mineral deficiency caused by dengue virus and strengthen the immune cells against it. 10 Moghadamtousi el al. (2014) reported that Curcuma Longa L. have antibacterial, antifungal and antiviral activities. This plant was found to be an inhibitor of HIV-1 and HIV-2 protease with IC 50 of 100 μM and 250 μM, respectively. 11 Aquoeus and methanol extract of Phyllanthus niruri L. (Meniran) which contains lignan, tannin, polyphenol, alkaloid, flavonoid, terpenoid, steroid, coumarin and saponin inhibit DENV-2 activity. 12,13 Tang et al. (2012) study on Andrographis paniculata (Sambiloto) and Cymbopogon citratus (Serai) based on cytophatic effect in vitro showed inhibition activity against DENV-1 75% and <50% respectively. 14 Previous studies showed that these plants have antibacterial activity, antiviral activity or both. [7][8][9][10][11][12][13][14] However, there is only limited study of these plants against dengue virus. This study aimed to know whether these plants have potential activity against dengue virus in vitro.

Natural Extract Preparation
The crude extract of leave of eight indigenous herbal plants as mention before were originated from Solo, East Java were extracted in ethanol and dried, and dissolved in dimethyl sulphoxide (DMSO) in Laboratory of The Faculty of Science, University of Sebelas Maret, Surakarta, Central Java. The leaves were obtained from the plants grew in Balai Besar Penelitian dan Pengembangan Tanaman Obat dan Obat Tradisional (B2P2TOOT) plantation in Tawangmangu, Karanganyar, Central Java. The extracts were protected from light and stored at -20°C. The stock solutions were then diluted in culture medium with the concentrations needed before conducting the assay.

Dengue Virus Propagation
DENV-2 strain NGC were obtained from National Institute of Health Research and Development, Ministry of Health Republic of Indoneisa with kind help of Dr. dr. Reni Herman, MBiomed and were propagated in Huh7it-1 cells in 2% FBS (Sigma, Aldrich, USA) DMEM (Gibco, Thermo Fisher Scientific, USA) medium at 37°C with 5% CO 2 for 5-7 days. Huh7it-1 cell propagation we used 10% FBS DMEM medium.

Dose-dependent assay
For dose dependent assay, we infected cells with DENV at a multiplicity of infection (MOI) of 0.5 FFU/cell. Firstly, the Huh7it-1 cells were prepared in 48 well-plate 5 x10 4 cell/well. Then, the extract was prepared with twice its concentration in a 96well round-bottomed plate. In a cold environment, afterwards, the virus was prepared with twice its MOI in the same 96-well plate and mixed with the prepared extract. Since there were no specific antiviral drugs to DENV as positive control, in this study we used medium and medium with DMSO (Sigma, Aldrich, USA) as positive and negative control. The mixture was then added to the Huh7it-1 cell after the medium was already removed. Next, incubation was done at 37°C with 5% CO 2 for 2 hours and agitated in every 30 minutes. Then, the mixture was disposed and the cell was rinsed two times with serum-free medium. After that, 3 wells of positive control and 3 well of negative control were prepared. Over-layer solution of natural extract was then added to the mixture. Afterwards, re-incubation was done for 48 hours. Lastly, the supernatant was harvested and stored in -80°C.

Cell Viability Assay
The cells were prepared in 96-well plate with 10% FBS DMEM medium and incubated for 24 hours. After the medium was removed, the extract solution was prepared with various concentrations. Controls DMSO at 0.3% and 0.1% as well as DMEM medium were put to the well plate with each 3 wells. Then, the prepared extract solution was added. The mixture was incubated for 48 hours. We added 10% MTT reagent (Sigma, Aldrich, USA) after removing the extract solution from each well. Re-incubation was done for 3 to 4 hours. Next, MTT reagent was discarded and DMSO 100% was added. Finally, the absorbance level was examined for further calculation of CC 50 .

Focus Assay
The Huh7it-1 cell was prepared in complete medium with 2.3 x 10 4 cell/mL concentration and placed in a 96-well plate for 24 hours. Stored supernatant was thawed first and serially diluted in Medium with 2% FBS. The huh7it-1 cell was then taken out from the incubator and the medium was removed. Next, the diluted supernatant was added into the cells. They were then incubated for 2 hours at 37°C with 5% CO 2 . The diluted supernatant was removed and overlaid with methylcellulose 0.5%. We incubated again at 37°C with 5% CO 2 for 2 days. Two days later the infected cells in each well were fixated using 200 μL 3.7% formaldehyde per well and incubated for 15 minutes, and the formaldehyde was removed after 15 minutes. After the formaldehyde was removed, the cells were washed three times with 200 μL of PBS with period of 5 minutes in between.
Immunostaining was then performed. After the PBS was removed, 100 μL of Tryton X 0.5% was added to each well. Afterwards, incubation was done for 30 minutes. The Tryton X was then removed from all wells. Next, the wells were rinsed with PBS 3 times. Then, 50 μL of 1/500 anti-dengue IgG from human serum was added. Incubation for 1 hour was then performed. After removing the antibody, each well was rinsed with PBS. After that, the second antibody, which was 50 μl of 1/500 human anti-IgG HRP (Sigma, Aldrich, USA), was added. It was incubated, removed, and rinsed again with the same method as before. Next, 100 μL of DAB (Thermo Scientific, USA) was added to each well. Last incubation was done for 5 to 15 minutes. Finally, the virus focus was observed under light microscope.

Statistical Analysis
The data was analyzed with unpaired t-test on GraphPad Prism 6. Normality tests of the data were performed using SPSS 21.

The effect of eight extracts to cell viability
The cytotoxic of eight natural extracts at 20μg/ mL were showed at Table 1. A herbal extract is considered to have in vitro activity based on three criteria, i.e cell viability >50%, IC 50 ≤25 µg/mL and selectivity index >3. [15][16][17] As shown in Table 1 the cell viability of all extracts were >50%, in other word all of extracts not toxic to the cells.

Screening of antiviral effect of eight extracts
As shown in Table 2, among the eight extracts tested P. guajava and C. papaya strongly inhibit virus replication (92.6% and 89.5% respectively) at 20 µg/ mL dose.

Cytotoxicity and antiviral activity of Psidium guajava
To ensure that extract was not toxic to the cell, the half cytotoxic concentration (CC 50 ) was measured by treating uninfected Huh7it-1 with various concentration of P. guajava, and cell viability was measured with MTT assay. The cell viability still showed a high level (almost 100%) after treated with P. guajava at concentration of 20 µg/mL. After treated with concentration more than 40 µg/mL, the viability decreased slightly. The CC 50 was analyzed from the linear regression equation of the percent viability. We found that the CC 50 of P. guajava was 153.18 μg/mL (Figure 1).
To determine antiviral effect of P. guajava, DENV was treated with various concentration of P. guajava. The infectivity decreased after treated with 40 µg/mL of P.
guajava. In the concentration of 80 μg/mL and 160 μg/ mL, we found no DENV infection. The IC 50 was analy zed from the linear regression equation of the percent infectivity. We found that IC 50 of P. guajava was 7.2 μg/mL ( Figure 2). The selectivity index of P. guajava was 21.28.

Cytotoxicity and antiviral activity of Carica papaya.
From MTT assay we calculated the viability of cells and compared with viability after treated with DMSO. The viability showed that treatment with C. papaya at concentration up to 40 μg/mL was not toxic to the cells, while the cell viability decreased at treatment with 80 μg/mL. From the linear regression equation of the percent viability, we found that the CC 50 of C. papaya was 244.76 μg/mL ( Figure 3).    Similar with P. guajava, we also measured infectivity of DENV-2 after treated with C. papaya. Addition of C papaya at the concentration of 40 μg/ mL and more showed 100% inhibition to DENV replication. Lower doses such as 20 μg/mL, 10 and 5 μg/mL showed percentage of infectivity at the level of 3.7%, 25% and 48% respectively. From the linear regression equation of the percent infectivity, IC 50 of C. papaya was 6.57 μg/mL (Figure 4). We found the selectivity index of C. papaya was 37.25.
papaya have inhibition activity 92.6% and 89.5% respectively, while the other extracts showed only inhibition activity less than 80%. All extract didn't show toxicity, i.e. cell viability >50%. These results suggest that P. guajava and C. papaya have good antiviral activity in vitro. This result is in agreement with study by Joseph et al. (2015), which also show that C. papaya leave chloroform extract inhibit DENV-2 in LLC-MK2 cells with CC 50 >1 mg/mL and EC 50 >1 mg/mL. 18 The extract of Psidium guajava was proven as an effective antiviral for dengue virus in vitro, might be caused this extract contained quarcetin (derivate of flavonoid) that inhibit the activity of reverse transcriptase enzyme. 19  These results are different from previous report that suggests antiviral activity of these plant extracts against dengue virus. Aqueous and methanol extract of P. niruri L. inhibits viral absorption into cells and inhibit DENV-2 replication. 13 However, study of P. niruri L. effect as antivirus is still limited. Methanol extract of C. longa was shown to interfere dengue virus replication by inhibiting NS2B/NS3 protease. 22 The different results might be caused by different geographic source of the plants. Plants from different geographic conditions may adapt to environment and produce different secondary metabolites. Flavonoid, tannin, alkaloid and saponin content increase following increase of altitude of the region where it grows. This increase may be aimed to protect plants from stressor such as UV-B and low temperature. 23 Different results in this study may also caused by different dose used in screening test. We used 20 μg/ mL, while Lee et al reports that the dose needed to inhibit 100% DENV2 replication is 250 μg/mL. 13 Our results showed that DENV-2 inhibition of   In conclusion, from screening antiviral activity of leave crude extract of eight indigenous herbal plants originated from Solo, East Java, P. guajava and C. papaya have potential antiviral activity against dengue virus in vitro. Further study is needed to confirm antiviral activity in vivo.