Ficus septica plant extracts for treating Dengue virus in vitro

Dengue virus types 1-4 (DENV-1-4) are positive-strand RNA viruses with an envelope that belongs to the Flaviviridae. DENV infection threatens human health worldwide. However, other than supportive treatments, no specific therapy is available for the infection. In order to discover novel medicine against DENV, we tested 59 crude extracts, without cytotoxicity, from 23 plants in vitro; immunofluorescence assay revealed that the methanol extracts of fruit, heartwood, leaves and stem from Ficus septica Burm. f. had a promising anti-DENV-1 and DENV-2 effect. However, infection with the non-envelope picornavirus, Aichi virus, was not inhibited by treatment with F. septica extracts. F. septica may be a candidate antiviral drug against an enveloped virus such as DENV.


INTRODUCTION
Dengue fever is an acute infectious disease caused by dengue virus (DENV), which is transmitted by mosquitoes to humans; about 50 million people are infected per year worldwide (Guzman et al., 2010). According to different serotypes of the virus, four types (DENV types 1-4) are divided. Each type has the ability to cause disease. DENV infection causes varying degrees of disease manifestation, such as self-limited febrile dengue fever, skin rash or drowsiness, agitation, liver enlargement, or dengue hemorrhagic fever (DHF) and even death. A second DENV infection may lead a life-threatening dengue shock syndrome (DSS) (Abel, Liautaud & Cabie, 2012;Kyle & Harris, 2008;Martina, Koraka & Osterhaus, 2009). Currently, no specific therapy is available for the infection other than supportive treatments (Guzman et al., 2010).
The identification and use of medicinal plants for treatment of various diseases has been done throughout human history. Certain medicinal plants also show antiviral activity, such as Carissa edulis Vahl against herpes simplex virus, Geranium sanguineum L. against influenza virus A, Boehmeria nivea L. against hepatitis B virus, Saxifraga melanocentra Engl. & Irmsch. against hepatitis C virus, Lycoris radiata (L'Hér.) Herb. against severe acute respiratory syndrome-associated coronavirus and Phyllanthus amarus Schum. & Thonn. against HIV (Mukhtar et al., 2008). In addition, the neem (Azadirachta indica A. Juss.) showed potential inhibition of DENV-2 replication (Parida et al., 2002). Thus, discovering a novel antiviral medicine from medical plants would be a promising strategy.
In this study, we collected 23 plants from among Taiwanese folk medicinal plants for screening anti-DENV herbs. We also investigated the antiviral effect on Aichi virus (AiV), a pathogenic picornavirus that causes gastroenteritis. Among these plants, we found that F. septica Burm. f. could be a potential medicinal plant against DENV.

Plant extracts
Materials of plant species were ground, extracted with the indicated solvent for one week. The extracts were concentrated under vacuum. Plant materials of the species no. 13 (leaves) was subjected to an additional one-step extraction with ethyl acetate (EtOAc) and filtered and dried as described before. The volumes (5 ml) of solvents were used per gram of plant material.

Cell proliferation assay
WST-1 assay (Roche, Basel, Switzerland) was used to monitor cell proliferation (Chou et al., 2014); cells were trypsinized and resuspended in culture medium, then plated at 5×10 3 cells per well in 96-well plates and incubated overnight. After plant extracts treatment for 48 h, the cells were incubated with 10 µl WST-1 reagent for 2 h. The cell viability was quantified by multi-well spectrophotometry (Anthos, Biochrom, Cambridge, UK). The absorbance at 450 nm was monitored, and the reference wavelength was set at 620 nm.

Treatment
In the extracts screening of DENV inhibition, the cells (5×10 3 cells) were treated with plant extracts with serial dilution dose of 100, 50, 25, 12.5, 6.25, 3.125 or 1.56 µg/ml or DMSO solvent control for 3 h. Then, these cells were infected by DENV-1, DENV-2 and AiV infection (multiplicity of infection [MOI] = 2.5). After 2 h virus adsorption, the medium mixture was replaced by fresh growth medium. At 42 h after infection, the virus-infected cells were analyzed by immunofluorescence assay. In another experiment, the viral stocks of DENV-1, DENV-2 and AiV were preincubated with a series of dilution doses of leaf methanol extract of F. septica (FS-(L)-M) for 1 h at room temperature. The mixture of virus plus the plant extract was then used to infect A549 cells.

Immunofluorescence assay
Immunofluorescence assay was conducted to determine the DENV and AiV infectivity as we previously described (Chen et al., 2013;Wang et al., 2015). In brief, cells were fixed with 4% paraformaldehyde for 30 min, then permeabilized with 0.5% Triton X-100 for 10 min. After two washes with phosphate buffered saline (PBS), cells were blocked with 10% skim milk in PBS. The cells infected with DENV or AiV were detected by antibody against NS3 (Yao-Houng, Biotechnology, Taipei) or anti-AiV VP1 antibody followed by IRDye 800 CM goat anti-mouse or -rabbit IgG (Li-Cor, Lincoln, NE, USA) or Alexa fluor 488 conjugated anti-mouse IgG (Thermo Fisher, Waltham, MA, USA) . The fluorescence intensity was measured and quantified by using the Li-Cor odyssey CLx imaging system or fluorescence microscopy (Zeiss, AX10).

Statistical analysis
Significant differences between groups were analyzed by 2 tailed Student t test with the software GraphPad Prism 6 (La Jolla, CA, USA). Data are presented as mean ± SD. P < 0.05 was considered statistically significant. The statistical datasets are showed in the supplementary information.

RESULTS AND DISCUSSION
We aimed to reveal a medicinal plant candidate against DENV. We extracted 70 different crude compounds from materials of 24 plants. DENV-caused respiratory disease was revealed (Rodrigues et al., 2014;Wang et al., 2007), in addition, our previous study showed that the lung carcinoma A549 cells were well susceptible target cells for DENV, these cells have been used in the study model of viral-host interaction. Thus, A549 cells were applied as the screening model in this study (Chang, Liao & Lin, 2006). The cytotoxic effect of the extracts was evaluated in lung carcinoma A549 cells by WST-1 cell proliferation assay. Except S. divaricatus, extracts from other 23 plants revealed no cytotoxicity effect at the maximum tested concentration, 100 µg/ml. Therefore, S. divaricatus extracts were excluded in the antiviral screening (Supplementary information, Table S1).  (Table 1). In addition, leaf extracts of F. sarmentosa var. henryi showed an anti-DENV-2 effect, with IC 50 72.04±14.5 µg/ml, which was higher than for the extracts of F. septica (Table 1).
In order to understand whether F. septica leaf extracted with methanol, FS-(L)-M, directly inactivated DENV, we preincubated viral stocks of DENV-1, DENV-2 and AiV with a series of dilution doses of FS-(L)-M for 1 h at room temperature. After virus adsorption, the mixture of virus plus the plant extract was replaced by fresh growth medium. At 42 h after infection, the virus-infected cells were analyzed by immunofluorescence assay      h at room temperature before used to infect A549 cells (3 × 10 4 ) at MOI = 2.5. After 2 h of virus adsorption, the virus-compound mixture medium was replaced by fresh growth medium for further 42 h incubation. DENV-or AiVinfected cells were detected by immunofluorescence assay. (B) Fluorescence intensity was measured and quantified by the Li-Cor odyssey CLx imaging system. The data are mean ± SD from three independent experiments. *** P < 0.001 by two-tailed Student t test.
Here, we reveal a new bioactivity of F. septica against dengue virus. Importantly, an enveloped virus but not non-enveloped virus is sensitive to the extract pretreatment, which suggests that certain compounds of F. septica might disrupt the DENV envelope structure or interfere with DENV contacting cells. Moreover, the anti-DENV effect of F. septica was demonstrated in the lung and liver cell types with clinical relevant.
F. septica may be a promising medical plant against DENV. The F. septica materials root bark acetone and leaf methanol extracts showed the best anti-DENV efficacy, further identification of the antiviral compounds from these two parts of F. septica would be important for drug development.