Antiviral activities of curcumin and 6‐gingerol against infection of four dengue virus serotypes in A549 human cell line in vitro

Dengue virus (DENV) is the most geographically widespread arbovirus causing dengue disease epidemics in tropical and subtropical regions. Nature provides abundant plants as a source for lead molecules against various diseases including DENV infection. We investigated the antiviral effect of curcumin and 6‐gingerol, the major active constituent of turmeric (Curcuma longa Linn.) and ginger (Zingiber officinale Roscoe), respectively, against all four serotypes of DENV infecting human lung epithelial carcinoma (A549) cell line in vitro. Both compounds generated cell cytotoxicity to A549 cells at CC50 values of 108 μM for curcumin and 210 μM for 6‐gingerol. The compound curcumin showed antiviral properties as described by IC50 of 20.60, 13.95, 25.54, and 12.35 μM, while 6‐gingerol of 14.70, 14.17, 78.76, and 112.84 μM for DENV‐1, ‐2, ‐3, and ‐4, respectively. Different levels of antiviral properties were observed between DENV serotypes. Our findings suggest that the antiviral assay of compounds against DENV should be performed to all four serotypes and not limited to a particular serotype. In conclusion, curcumin and 6‐gingerol exhibit antiviral properties against DENV infection and could provide a new therapeutic approach for dengue disease treatment strategies.


Introduction
Dengue virus (DENV) is considered the most geographi cally widespread arthropodborne virus with an increased frequency of epidemics and severe disease manifestation in hyperendemic countries (Guzman and Harris 2015). It is estimated that 390 million cases of DENV infections oc cur worldwide annually (Bhatt et al. 2013; Guzman andHarris 2015). Over the past 50 years, the world has ex perienced a dramatic increase in dengue incidence to al most 30folds (Guo et al. 2017), facilitated by virus trans mission predominantly by Aedes aegypti and to a lesser extent Aedes albopticus mosquito vectors (Guzman et al. 2016). The four DENV serotypes (DENV1, 2, 3, and 4) can cause a wide range of disease in human, from asymptomatic, classical fever Dengue Fever, to the life threatening conditions Dengue Hemorrhagic Fever and Dengue Shock Syndrome (Martina et al. 2009).
The current disease management for DENV infection is limited to early detection, fluid replacement, and symp tomatic therapy, although research has proven the associ ation of viral load with the severity of the disease in the patients (Wang et al. 2003). The firstever dengue vaccine Dengvaxia (CYDTDV) still has limited use in the target population below nine years old (Dighe et al. 2019) and the development of antiDENV drugs has been in a slow process. There is no licensed antiviral therapy available to date and only four small molecule antiDENV drugs have entered phase I and phase II clinical trials. Although these drugs meet the required safety profile, they could not reduce viral load as expected (Tian et al. 2018). With the current unavailability of antiviral therapy for DENV infection, a search for compounds having antiviral effect needs to be established. One of the possible sources of a compound with antiDENV activities will be from natural sources.
Human alveolar epithelial A549 cells have been proven suitable for use as a DENV infection model in vitro (Yohan et al. 2014). The use of A549 cell line in DENV research was based on the evidence of viral anti gen detection in macrophages and vascular endothelial cells of the lung (Jessie et al. 2004). The antiviral ef fect of curcumin and 6gingerol to DENV has been re ported although limited to a particular serotype (Padilla S et al. 2014; Sharma et al. 2015. The differing genetic of DENV serotypes may cause differences in their viru lence (Holmes and Burch 2000). Hence, an antiviral study against all four DENV serotypes in human cells will give more insights into dengue pathogenesis. In this study, we investigated the antiviral effect of curcumin and 6gingerol on the growth of all four DENV serotypes in human A549 cell line. Findings from this study may be beneficial for the development of antiDENV drug therapy from natural resources as alternative therapeutic approach for dengue disease.

DENV serotypes
Four DENV serotypes of Indonesian isolates were used. All virus stocks were generated from a lowpassage num ber (maximum of five passages) of the virus in Vero cells. The DENV1 strain JMB034 was isolated from a dengue patient in Jambi (Haryanto et al. 2016). The other serotypes DENV2 strain SUB011, DENV3 strain SUB 006, and DENV4 strain SUB007 were isolated from pa tients in Surabaya, East Java (Aryati et al. 2013; Ward hani et al. 2017). The harvested culture supernatants con taining viable DENV were stored at 80°C until use and their titers were measured using standard plaque assay method in BHK21 cells, as described previously (Yohan et al. 2014; Sasmono et al. 2015.

A549 cell viability test
Curcumin (≥ 65%, HPLC) and 6gingerol (≥ 98%, HPLC) were purchased from SigmaAldrich in powder and lyophilized forms, respectively. Both compounds were solubilized using dimethyl sulfoxide (DMSO) (Sigma Aldrich, USA) as a vehicle into stock solutions of known molarity. The stock solutions were then diluted into work ing solutions using RPMI medium supplemented with 10% FBS. The cytotoxicity of the compounds was tested in A549 cells, seeded 1×10 5 cells/well of 96well plate and grown overnight at 37°C, 5% CO 2 . Serially diluted com pounds were added into the cells monolayer in triplicate and tested with 3(4,5dimethylthiazol2yl)2,5diphenyl tetrazolium bromide (MTT) using the MTT Cell Prolifer ation Assay Kit (Trevigen, Gaithersburg, MD, USA) after 48 h of incubation at 37°C, 5% CO 2 , according to the protocol described by the manufacturer. Following the formation of formazan complexes, the plate was read at 570 nm using a microplate reader. The percentage of cell viability was measured as a comparison to the medium only controls. A doseresponse curve was obtained using nonlinear regression (curve fit) and the cytotoxic concen tration was calculated as median cell cytotoxicity (CC 50 ) in which the concentration of compound that reduced the cell's viability by half.

DENV antiviral activity testing
The A549 cells were seeded at 1×10 5 cells/well in 96well plate and subjected to infection with DENV1, DENV2, DENV3, or DENV4 with multiplicity of infection (MOI) value of 1, described hypothetically as one virus particle per cell. Compounds treatment was done using 10, 25, and 50 μM of curcumin and 50, 100, and 200 μM of 6 gingerol, selected as subcytotoxic concentrations follow ing the CC 50 values determined from A549 cell viability test. Treatment of cells with calculated concentrations of compounds was done using cotreatment method, where the mixture of DENV and compounds was added to cell monolayer and allowed to react at 37°C, 5% CO 2 for 48 h, without removal of inoculant or wash steps, as described previously (Chen et al. 2013). Following the incubation period, the supernatant was transferred into microtubes for the virus titration using plaque assay in BHK21 cells. The calculated virus titer was normalized to the medium only control (without compound or 0 µM) and presented as a percent of inhibition. The median inhibitory concentration (IC 50 ) was measured as the concentration of compound that can inhibit 50% of virus titer. A vehicle control is used to monitor the effect of DMSO diluent in the system. The vehicle control was prepared as 0.1% v/v of DMSO, a concentration that was higher than the highest concentra tion of compounds in the testing.

Statistical analysis, CC 50 and IC 50 calculations
Statistical analyses were conducted using SPSS Statis tics software version 17.0 (SPSS Inc., USA). Mean and standard error of mean (SEM) from three independent experiments were calculated. The comparison between groups in experimental results was analyzed using one way ANOVA, followed by Tukey's post hoc test. A p value less than 0.05 was considered as statistically signifi cant. The CC 50 and IC 50 values were calculated using AAT Bioquest Quest Graph IC 50 calculator (available online at https://www.aatbio.com/tools/ic50calculator) based on the fourparameters logistic equation to generate a sigmoid function.

Cytotoxicity of curcumin and 6-Gingerol to A549 cells
The cells' cytotoxicity properties of curcumin and 6 gingerol were measured using standard MTT assay for measurement of cells' viability. Both curcumin and 6 gingerol reduced the viability of A549 cells in a dose de pendent manner. The median cell cytotoxicity CC 50 of curcumin and 6gingerol were measured at 108 μM and 210 μM, respectively (Figure 1). Both curcumin and 6

Antiviral activities of curcumin and 6-gingerol against DENV
Following the CC 50 data, the antiviral testing of cur cumin and 6gingerol was set at subcytotoxic concentra tions. Curcumin and 6gingerol significantly inhibited the growth of all DENV serotypes (Figure 2 and 3, respec tively). For curcumin, all concentrations tested signifi cantly inhibited the growth of all DENV serotypes com pared to the mediumonly group (p < 0.05), except in two experimental conditions at 10 µM and 25 µM in DENV3 (Figure 2). On the other hand, for 6gingerol, all concen trations tested show a significant reduction in the growth of all DENV serotypes (p < 0.05) except at 50 µM in DENV3 ( Figure 3).
The inhibition profiles were increased along with the increasing concentration of compounds compared to the medium only control. Vehicle control resulted in not sig nificant reduction of virus titers compared to control (data not shown). We observed a variable median inhibitory concentration IC 50 results for each DENV serotype ( Table  1).

Discussion
The significant increase in dengue research during the past decades covers topics on dengue virology, pathogenesis, and immunology and progress in developing antivirals, vaccines, and new vectorcontrol strategies that are impor tant for dengue control and prevention (Guzman and Har ris 2015). We tested the antiviral properties of curcumin and 6gingerol against all four serotypes of wild strain DENV on A549 cell lines. The A549 cell line was used for its susceptibility to DENV infection and showed supe rior suitability compared to other human cell lines (Yohan et al. 2014).
As an active compound from nature, curcumin and 6 gingerol can also be toxic and induce apoptosis to cells (Kaushik et al. 2012; Impheng et al. 2015. We found that curcumin has cytotoxic property although it did not signif icantly affect A549 cells' viability in concentrations up to 50 μM with CC 50 of 108 μM after 48 h of cell treatment (Figure 1a), a result that is comparable to a previous study by Kaushik et al. (2012). In the other hand, 6gingerol showed cytotoxicity to A549 cells with CC 50 of 210 μM (Figure 1b), comparable to other result observed elsewhere (Kim et al. 2008).
Antiviral activities of curcumin against DENV2 has been reported (PadillaS et al. 2014; Balasubramanian et al. 2019. The activity against DENV or enveloped virus is believed through the inhibition of Ubiquitin Pro teasome System (UPS) (Chen et al. 2013; PadillaS et al.  (Glickman and Ciechanover 2002). In this study, we found that curcumin could inhibit all four DENV serotypes in the same manner (Figure 2), although different levels of IC 50 was observed for each serotype (Table 1). Our result suggested similar antiviral potency of curcumin to all DENV serotypes. The effect of curcumin to DENV2 was in accordance with pre vious result by PadillaS et al. (2014). To the best of our knowledge, this is the first study to explore the antiviral ability of curcumin against all four serotypes of wildtype DENVs. The possible mechanisms of curcumin's antiviral properties include membranedisturbing properties (Chen et al. 2013), altering membrane fluidity (Anggakusuma et al. 2014), and inhibiting cell binding (Mounce et al. 2017).

2014) which plays a role in viral replication
The compound 6gingerol is known to exhibit a va riety of biological activities including anticancer, anti inflammation, and antioxidant (Wang et al. 2014). In this study, 6gingerol, also revealed antiDENV proper ties shown by the reduction of viral growth along with the increasing 6gingerol concentration in the in vitro sys tem (Figure 3). Previous study demonstrated the ability of fresh lipophilic juice of ginger to cause complete inhi bition of HCV replication, a member of Flavivirus family like DENV (Eladawi et al. 2011). Fresh ginger is effective against HRSVinduced plaque formation on HepG2 and A549 cell lines by blocking viral attachment and internal ization by interfering G protein and F protein (Chang et al. 2013). The administration of 6gingerol was manifested as inhibition of fatty acid synthase (FASN) expression (Im pheng et al. 2015), which is a biosynthetic pathway to es tablish DENV replication complexes (Heaton et al. 2010). The aqueous extract of ginger rhizome inhibited the ac tivity of matrix metalloproteinase (MMP)2 and MMP9, while upregulating the expression of tissue inhibitor met alloproteinase (TIMP)1 and TIMP2 in DENV infected cells (Sharma et al. 2015). Interestingly, the antiviral ac tivity of 6gingerol was found to be higher in DENV1 and 2 compared to DENV3 and 4. The fact that 6gingerol has different antiviral properties to four DENV serotypes is of merit for further confirmatory exploration whether different genetic of DENV serotypes attributed to these findings.
Our results add information on the antiviral proper ties of curcumin and 6gingerol against all four serotypes of DENV. In addition, we promote that antiviral assay of compounds against DENV should be performed to all four serotypes and not limited to a particular serotype. Never theless, our study has limitations. The study design was aimed only to inspect the antiviral properties of curcumin and 6gingerol in four DENV serotypes. The biological pathway mechanisms underlying the virus inhibition ef fect were not sought. More indepth studies are needed to confirm our findings. In addition, the availability of different compound sources with different purities in the market may also give impact on the results generated. To the best of our knowledge, this is the first study describing the antiviral effects of curcumin and 6gingerol to all four DENV serotypes.

Conclusions
In conclusion, curcumin, the major active compound of turmeric and 6gingerol, the major active constituents of ginger, have shown antiviral activities against all four serotypes of DENV. Curcumin and 6gingerol may have potentials in the development of antiDENV drug therapy from natural resources and could provide an alternative therapeutic approach for dengue disease treatment strate gies.