Drug of Action Cassia Alata Leaves Extract as Antiviral to Dengue Virus Serotype-2 In vitro

Dengue is recently reported to be the most rapidly spreading mosquito-borne viral disease and is endemic in more than 110 countries including Indonesia with two-fifths of the world population at risk. According to the data from Ministry of Health Republic Indonesia, more than 100 000 people were diagnosed with dengue fever in the 2014 and were found to be fatal in almost 1000 cases.1 An estimated 100 million cases of dengue fever and half a million cases of dengue haemorrhagic fever (DHF) occurred annually.2 The clinical presentations of DENV infections range from asymptomatic to severe illness that may lead to death if improperly managed. The symptomatic cases are categorized as undifferentiated febrile illness (UF), dengue fever (DF), dengue hemorrhagic fever (DHF), dengue shock syndrome (DSS) and unusual dengue (UD) or expanded dengue syndrome (EDS).3


INTRODUCTION
Dengue is recently reported to be the most rapidly spreading mosquito-borne viral disease and is endemic in more than 110 countries including Indonesia with two-fifths of the world population at risk. According to the data from Ministry of Health Republic Indonesia, more than 100 000 people were diagnosed with dengue fever in the 2014 and were found to be fatal in almost 1000 cases. 1 An estimated 100 million cases of dengue fever and half a million cases of dengue haemorrhagic fever (DHF) occurred annually. 2 The clinical presentations of DENV infections range from asymptomatic to severe illness that may lead to death if improperly managed. The symptomatic cases are categorized as undifferentiated febrile illness (UF), dengue fever (DF), dengue hemorrhagic fever (DHF), dengue shock syndrome (DSS) and unusual dengue (UD) or expanded dengue syndrome (EDS). 3 The disease is caused by dengue virus (DENV) which exists as four closely related, but antigenically distinct virus serotypes of DENV-1 to DENV-2. 2 This leads to many challenges in the development of a vaccine that would provide solid immunity equally to all serotypes. 3 Another approach of viral diseases control is directed toward antiviral against DENV. During several decades a research has been explored natural products from medicinal plants due to the advantages of high chemical diversity of these biologically active molecules and lower cost. Many plant composition such as flavonoids, terpenoids, lignin, alkaloids, tannins, polyphenolics, coumarins, saponins, and chlorophyllins, are reported have anitival activity to DENV. 4-6 Therefore, natural products from herbal medicine represent an alternative approach for treatment of DENV infections. Research for dengue antiviral has focused on the different phases of the viral lifecycle (virus attachment, viral entry, trafficking, translation or replication).
Nevertheless, only few antiviral therapies have been tested and little is known about the effects and mechanisms of the antiviral agents. 4 Our study previously reported inhibitory activity of C. alata extract against DENV-2 and its toxicity to Huh-7 it-1 cells, but the inhibitory mechanism and chemical content of C. alata is already unknown. 7 This study aims to evaluate drug of action Cassia alata leaves extract and its fraction as antiviral DENV-2 using cell culture model and knowing the compounds contained in Cassia alata.

Study design
This study was an experimental study using Huh7 it-1 cell line for pursuing the DENV antiviral mechanism. Compounds identification of C. alata was performed using LC-MS / MS.

Preparation of extrac
The boanical identities of Cassia alata leaves extract was determined and authenticated by the Botanical in Research Center for Biology LIPI. After the taxonomy identification, the Cassia alata leaves were washed and cleaned prior to air drying at room temperature. Dried and ground leaves (1000 g) were submitted to extraction at room temperature with ethanol 70%. The solvent was removed used vaccum evaporator yielding 225.7 g of the thick extract. The extract (60 g) was initially partitioned with n-hexane, ethyl acetate, butanol and water. The solvent was removed used vacuum evaporator yielding 4.2 ; 7.7 ; 7.1 and 31.5 g respectivelyExtracts and fractions were diluted at concentration of 100 mg/ml in dimethyl sulfoxide (DMSO) (Sigma Aldrich, USA) as a stock solution. 7 Identification compound of C. alata extract using LC/ MS-MS Mass spectrometry was performed on a Xevo, G2-XS QTof (Waters MS Technologies). Ionisation type is ESI. The scan range was from 100 to 1200 m/z. The capillary and cone voltage was set at 0.8 kV and 30 kV, respectively and was used positive electro spray mode. The desolvation gas was set to 1000 L/h at a temperature of 500°C and the cone gas was set to 50 L/h and the source temperature was set to 120°C. The UPLC analysis was performed using a Waters Acquity Ultra Performance LC system. Chromatographic separation was carried out on an ACQUITY UPLC HSS T3 C18 column (100 mm x 2.1 mm, 1.7 µm) at a column temperature of 40 °C. The mobile phase consisted of solvent A (0.1% formic acid in water, v/v) and solvent B (0,1% formic acid in acetonitrile), with gradient polarity from 95:0.5 (A: B) to 0.5:95 (A: B). The flow rate was set at 0.3 mL/min. The column and auto sampler were maintained at 40 °C and 20 °C, respectively. The injection volume was 1 µL.The data acquisition and processing were performed using UNIFI. The parameter used was retention time (RT) in the range of 1-15 min. Determination of the dominant compound was carried out with semi quantitative determination based on the high intensity of the compound on the LC-MS/MS graph.

Preparation of DENV-2 and Huh-7-it-1
We used DENV serotype 2 strain New Guinea C (DENV-2 NGC) adapted in Huh-7-it-1 cell line. A monolayer of Huh-7-it-1 cell in T-75 flasks were infected with DENV-2 NGC with moi of 0.5 PFU/cell and incubate at 37 o C with 5% CO 2 for 7 days. During the time of virus propagation, the FBS concentration of the cell culture medium was reduced to 2%. Supernatant was harvested and centrifuged at 1000 g for 5 minutes. Culture supernatant was stored at -80 o C and checked for the titer of dengue virus by Focus assay. 8,9 Determination of cytotoxicity In vitro cytotoxicity was determined by MTT assay based on viability of Huh-7-it-1 after treated with extract. In 48 well flat-bottom plates (Corning, USA), we added 5 × 10 4 cells/well and incubated at 37 o C, 5% CO 2 for 24 hours. After 24 hours, the cells were treated with various concentration of extract from 0.1 to 80 g/mL and incubated at 37 o C, 5% CO 2 . After 48 hours, 20 μL of 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) (Promega) salt solution was added into each well and incubated for 4 hours according to the manufacturer's instruction. The absorbance reading of each well was measured using micro plate reader at 490 nm. 7 The percentage of cell viability and toxicity was further determined based on the absorbance readings. First, we calculate the theoretical percentage toxicity of the samples by dividing the mean blanked sample ODs by the mean blanked control ODs for each sample. We used the viability data correspond to the cytotoxic effect. 8,10 Determination mechanism of antiviral action of CA and its fractions To determine mechanism of antiviral action that would explain the possible inhibitory step(s) in the viral life cycle, we used published method with slight modification. 11,12 We carried out a time-of-addition study with the concentration of CA and it's fractions of 10 mg/mL and monolayer (5×10 4 cells/well) Huh-7-it-1 at 48 well plate.
For inhibition of DENV receptor (i), we added extract or fractions into cell for 2 hours before DENV infection at 37 o C with 5% CO 2 , The residual extract or fraction were removed and washed. Then DENV-2 was infected into cells for for 2 hours at 37 o C with 5% CO 2 . After virus adsorption , the remain virus was removed and the medium without extract was added and incubated for 48 hours. For entry step experiment (ii), DENV-2 was mixed with extract or fractions at concentration of 10 ug/mL, then infected to the cell for 2 hours at 37 o C with 5% CO 2 . After virus adsorption, the residual virus and test sample were removed and washed . Then the cells were added with medium and incubated at 37 o C with 5% CO 2 for 48 hours. Post entry step experiment (iii), DENV-2 was infected to the cells for 2 hours, and removed the residual virus. The cells were added with medium containing of Extract and its's fraction and incubated for 48 hours. The whole step experiment or prepost experiment (iv) was combaining of pre and post experiment. The DENV-2 were treated with exctrat or it's fraction prior infection and followed by addition of medium with medium containing of extract and its's fraction As a positive control: we infected cells with DENV-2 and the cells were added with medium without extract of it's fraction for 48 hr.
After 48 hours, then we harvest and determine virus titer by focus assay with the same method above. 8,11 . The result from focus assay was used to determine percentage of virus inhibition. 8,13

Cytotoxicity of C. alata extract and fractions on the Huh7 it-1 cell line
Based on previous tests it was known that CA, CA1, CA2, CA3, CA4 had DENV antiviral activity on Huh 7-it-1 cell with the focus assay method, where there was a decrease in the focus on dengue-infected Huh 7-it-1 cells. 7 Moreover, we want to investigate antiviral DENV mechanisms from extracts of CA, CA1, CA2, CA3, and CA4 through time addition studies. To be a potential antiviral drug the extract not only has to be able to inhibit growth of the virus, but also remain harmless to the cells. Therefore, we determine the toxicity after the cells were treated with extact and it's fraction at concentration of 10 µg/mL using MTT assay. CA, CA1, CA2, CA3 and CA4 showed no toxic effect to the cell based on the percentage of viability cell of Huh7 it-1 ( Table 2).

Time of addition studies cassia alata leaves extract and fractions
Dengue virus (DENV) infection begins with the attachment of a viral capsid protein to the receptor (attachment) which in turn mediates endocytosis through the formation of endosomes with low pH and induces fusion of DENV. 21,22 This stage can be a target of antivirals where obstacles to the viral envelope or parts of cell receptors will prevent infection. In this study the resistance to virus particles was directly tested by exposing CA, CA1, CA2, CA3, CA4 to DENV-2 isolates, then inoculating the cells. The result of viral titers at the addition of CA at a dose of 10 µg/ml, inhibition reached 99.28%. While the virus inhibition after the addition of CA1, CA2, CA3, CA4 were 92, 63, 85 and 89%, respectively. CA has the most potent activity in damaging the integrity of membranes on the surface of virus particles. The next test is the addition of CA, CA1, CA2, CA3, CA4 extract to the medium after the cell is infected. The test results provide inhibition of 99.06% at the concentration of CA 10 µg/ml. Whereas for fractions of CA1, CA2, CA3 and CA4 fractions give inhibitions of 100, 100, 88, 87% respectively ( Figure 6).
The non-polar fraction of hexane (CA3) and ethyl acetate (CA2) gave better results in the post-infection mechanism. It was estimated that the content of non-polar compounds found in the fraction of CA2 were an active compouds to inhibit DENV. Analysis with LC-MS / MS, known that the CA2 and CA3 contain several same compounds i.e. ,7,2 ', 5'-Tetrahidrox-flavone, Daturametelin H and Kaempferol-3,7diglukoside. In this mechanism, after infection, antiviral is continuously present in the culture medium. In a previous study reported tests on vero cells added with medium and curcumin and incubated for one day showed that curcumin can accumulate in cells when detected using a confocal microscope. 23 This result indicates CA, CA1, CA2, CA3, CA4 can inhibit DENV replication in cells. CA is an ethanol extract that is semipolar in nature, presumably the nonpolar part of CA which is lipophilic can penetrate the cell membrane. 22 Replication of the viral genome occurs Angelina, et    in the cytoplasm, then towards the endoplasmic reticulum where viral particles are assembled, antiviral accumulation in the cell cytoplasm is thought to interfere with the DENV replication process. 24 In the treatment where exposure of CA, CA1, CA2, CA3, CA4 before infection to DENV-2 and the addition of CA to the medium after the infected cell gave a maximum barrier of 99.06%, whereas when compared to the results of inhibition at the attachment and postinfection stages CA was concluded to be better inhibited at the postinfection stage ( Figure 6).
While in the treatment of CA exposure to cells or receptor blocks, the inhibition percentage was 96.04% ( Figure 6). The possible inhibition mechanism of CA in this way is on the cell membrane by the accumulation of CA, as well as things related to replication in the cell.
These results indicate that to be able to inhibit DENV replication in cells exposed to CA for 2 hours, CA requires a concentration of 10 µg/ ml.
These studies demosntrated that ethanol extract of Cassia alata showed strong inhibition in both early step (receptor and attachment to host cells) and post infection with inhibition 96.04 % and 99.16 % ( Figure  6).Compared with those fractions, Cassia alata ethanol extract has strongest inhibition DENV in every step of virus replication. We supposed the antiviral activity of CA extract is influenced by the compounds contained in the extract. Kaempferol -3,7-diglukoside is the highest intensity compound contained in CA extracts compared to fractions. From the previous research, it was reported that kaempferol glycosides are good candidates as antiviral for 3a channel proteins of coronaviruses. 25 To know the mechanism more deepply especially which protein of DENV was inhibit by CA and fraction could be investigate through bioinformatic and enzymatic testing of each protein DENV.