Novel Antiviral Investigation of Annona squamosa Leaf Extract against the Dengue Virus Type-2: In Vitro Study

Dengue disease is a crucial mosquito-borne viral infectious disease in subtropical and tropical regions. Approximately, it reported about 350 million cases in global and more than 2.5 billion people are at high risk.1 Indonesia is a tropical country and it is common home of mosquito vector species of dengue virus (DENV), Aedes aegypti, and Aedes albopictus.2 The infection is caused by four DENV serotypes (DENV-1 to DENV-4) belonging to the Flaviviridae family.1 Up to now, there is no effective an antiviral available for this dengue disease.2,3 However, the vaccination now had been trialed in several endemic countries, including Indonesia.4


INTRODUCTION
Dengue disease is a crucial mosquito-borne viral infectious disease in subtropical and tropical regions. Approximately, it reported about 350 million cases in global and more than 2.5 billion people are at high risk. 1 Indonesia is a tropical country and it is common home of mosquito vector species of dengue virus (DENV), Aedes aegypti, and Aedes albopictus. 2 The infection is caused by four DENV serotypes (DENV-1 to DENV-4) belonging to the Flaviviridae family. 1 Up to now, there is no effective an antiviral available for this dengue disease. 2,3 However, the vaccination now had been trialed in several endemic countries, including Indonesia. 4 Indonesia is a huge country in Southeast Asia with high of flora diversity in the world. There are more than 5,000 medicinal plants that available all around us. 3 Consequently, medicinal plants are utilized by people for curing various diseases, including for viral related diseases. 3,5,6,7 Various medicinal plants were found as an antiviral compounds, 8 such as Amaryllidaceae, 9 Annonaceae, 10 Euphorbiaceae, 11 and many others.

Preparation of Annona squamosa leaf extract (ASLE) Preparation of Vero cells and DENV-2 isolate
The Vero cells was purchased from ATCC: The Global Bioresource Center, USA which cultured in medium containing 2% penicillinstreptomycin and 10% FBS (Gibco, USA) and incubated at 37 °C with 5% CO 2 . A confluent monolayer of Vero cells was detached with trypsin-EDTA and incubated at 37 °C for 5 minutes. Furthermore, medium was added, pipet gently, and counted by using a hemocytometer (Paul Marienfeld, Germany). Cells were cultured into 96-well plate with density 1×10 6 cells/10 mL and incubated at 37 °C with 5% CO 2 16 . In this study, we used DENV-2 which originally isolated from Surabaya, Indonesia. We used a clinical DENV-2 isolate identified by the Dengue Study Group, Institute of Tropical Diseases, Universitas Airlangga (NCBI accession number: KT012513). Previous study revealed that all DENV-2 strains isolated in Surabaya were classified into Cosmopolitan genotype. The clinical isolates propagated and maintained as previously described by Kotaki et al. (2016). 17 After titration of the virus isolate, the stocks were stored at -80 °C for further experiments.

Cytotoxicity assay
The confluent monolayers of Vero cells were prepared on a 96well plate (1×10 5 cells/mL) as previously described by Untoro et al. (2019). 18 We used CellTiter-Glo® Luminescent Cell Viability Assay (Promega, USA) in method to set the number of cells culture based on the ATP quantitation. Furthermore, CellTiter-Glo® Luminescent Cell Viability Assay was addressed for cytotoxicity test by following the manufacturer's guidelines.

Antiviral activity assay
The confluent monolayers of Vero cells were prepared on a 96-well plate (1×10 6 cells/10 mL), and the titer of DENV-2 was added in 2×10 4 FFU/ well. In this study, we used Viral ToxGlo™ Assay (Promega, USA) as a method intended to identify the cytopathic effect induced by a viral infection. In addition, we used Viral ToxGlo™ Assay reagents following the manufacturer's guidelines. Furthermore, the calculation of the selectivity index (SI) was defined as the comparison with ratio of 50% cytotoxic concentration (CC 50 ) and 50% antiviral concentration (IC 50 ) as previously stated by Zandi et al. (2012). 19

RESULTS AND DISCUSSION
Dengue is a mosquito-borne disease whose broad incidence has upward dramatically in the last ten years. 3,20 Indonesia is one of the greatest dengue-endemic nations in the worldwide which approximately 100,000 incidences of dengue are informed per annum. 17 The East Java, West Java, and Central Java are considered for being the most prevalent province for dengue. 21 Interestingly, in this study, we used DENV-2 isolate (NCBI accession number: KT012513) from the previous study conducted by Kotaki et al. (2016). 17 It had a higher ratio of illness severity as compared to the other serotypes, such as DENV-1, DENV-3, and DENV-4 in Brazil. 22 Recently, secondary infection with DENV-2, DENV-3, and DENV-4 increased the risk of severe dengue infection within Southeast Asia. Furthermore, DENV-2 in vitro proliferation was already established and standardized. 23 In addition, we rendered molecular phylogenetic modeling and tree visualization by applying Molecular Evolutionary Genetics Analysis X (MEGA X) software with the maximum likelihood method based on the envelope glycoprotein gene from Indonesia and other countries (Figure 1). The phylogenetic tree was validated by performing the analysis on 1000 bootstrapped input datasets and crossreferencing with the Tamura-Nei substitution model. 24 Nowadays, the use of traditional herbal medicines to manage various illness is accretion globally. 3,7,25,26,27 Medicinal plants are a potential source for the development of new antiviral drugs. The plants contained of various chemical composition with the potency to prevent viral replication and control the viral infection. 11 Plants have been considered to have an antiviral action and some have been accustomed to manage viral taints in humans and animals. In brief, the member of Annonaceae, Zingiberaceae, Cucurbitaceae, Fabaceae, Myrtaceae, Caricaceae, Meliaceae, Poaceae, Acanthaceae, Euphorbiaceae, Halymeniaceae, Piperaceae, and many other families have been reported as an anti-DENV. 8 However, up to now, there is no effective antiviral or vaccine available for the dengue disease. 2 The research revealed that ASLE inhibited the DENV-2 in Vero cells with IC 50 : 73.78 μg/mL, CC 50 : 331.54 c, and SI: 4.49 ( Figure 2). Furthermore, we tested the ASLE activity in various concentrations and observed that ASLE exhibited low cytotoxicity effect within all evaluated concentrations (viability >50%) (Figure 3). In detail, we used various extract concentrations, such as 200 μg/mL, 100 μg/mL, 50 μg/ mL, 25 μg/mL, 12.5 μg/mL, and 6.25 μg/mL. The treatment of ASLE showed that the reduction of DENV-2 replication is demonstrated by the lowest concentration of extract (6.25 µg/mL). Our findings showed that ASLE exhibited the consequential antiviral action against DENV-2 in Vero cells. Furthermore, this study also suggested the leading antiviral activity of ASLE is possible in consequence of its action towards the stages of intracellular replication of the virus in place of the early stages of its replication sequence such as virus entry. Nevertheless, the completed frameworks of the activity required to be discovered for an anti-DENV medication. Further investigation might be applied by using the suitable model, for example cultured human cells.
Annona is one of 129 genera of the Annonaceae family which contained of 119 species. Annona squamosa is commonly called by people  as srikaya (Indonesia) or sugar apple (English). 28 It is also known for its delicious sweet taste. 29 The Province of East Java, Indonesia is considered as the endemic of Annona squamosa even though it is widely distributed to all the regions. The fruit is marketed as family fruit for daily consumption or as a source of the other food products in the home industry. 30 On the other hand, phytochemical screening of ASLE showed the availability of acetogenins, alkaloids, flavonoids, glycosides, phenolic, saponins, steroids, tannins, terpenoids, and many other compounds. 6,10,29,31 Therefore, Annona squamosa considered as the medicinal plant for many diseases, such as cancer, cardiac disease, diabetes, mucous diarrhea, and many other diseases. 6,29 According to the ASLE, previous research revealed that the cytotoxic effect of chitosan-based nanoparticles of ASLE on cervical cancer cells (HeLa) had an IC 50 value with 344.48 μg/mL in concentration. 6 Even though, many studies on the biological effects of Annona squamosa were conducted, there are only two reports have shown Annona squamosa as an antiviral against HIV 31 and avian influenza virus. 10 Furthermore, Lisina and Piramanayagam (2014) also demonstrated its binding properties by the in silico study between the HIV protease and chemical compound of Annona squamosa. 31 Yunita et al. (2019) stated that the most effective concentration of Annona squamosa seeds extract is 0.35 μg/mL. The inhibition mechanism of the extract was performed by interfering the attachment and reducing the viral replication. The flavonoids were suspected to have a mechanism for interfering the viral replication by inhibit the viral reverse transcriptase enzymes which resulted in blockade synthesis of an virus RNA. 10 Moreover, compound which considered as a drug should have the cytotoxic activity only at high concentrations and activity at very low concentrations counter the virus. In brief, it consequently acquiescent a high SI rate and capable to eradicate the DENV at concentrations well below its cytotoxic concentration. 19 In addition, the SI of a compound is a highly fulfilled as a variable accustomed to declare the in vitro effectiveness toward the virus replication inhibition. Moreover, the establishment of new antiviral invention from bioactive composition is indispensable in pursuance of more potent and less toxic antiviral drugs. 16

CONCLUSION
In summary, ASLE demonstrated the antiviral activity against the DENV-2 with less toxicity, and high possibility to be considered as a drug candidate. Therefore, in vivo assessment might need to be conducted for further DENV-2 antiviral identification.

DISCLOSURE STATEMENT
The authors have no conflicts of interest to declare.