Antiviral Activity of Isatis indigotica Extract and Its Derived Indirubin against Japanese Encephalitis Virus

Isatis indigotica is widely used in Chinese Traditional Medicine for clinical treatment of virus infection, tumor, and inflammation, yet its antiviral activities remain unclear. This study probed antiviral activity of I. indigotica extract and its marker compounds against Japanese encephalitis virus (JEV). I. indigotica methanol extract, indigo, and indirubin proved less cytotoxic than other components, showing inhibitory effect (concentration-dependent) on JEV replication in vitro. Time-of-addition experiments proved the extract, indigo, and indirubin with potent antiviral effect by pretreatment (before infection) or simultaneous treatment (during infection), but not posttreatment (after entry). Antiviral action of these agents showed correlation with blocking virus attachment and exhibited potent virucidal activity. In particular, indirubin had strong protective ability in a mouse model with lethal JEV challenge. The study could yield anti-JEV agents.

Japanese encephalitis virus (JEV) belongs to genus Flavivirus of the Flaviviridae family, an arthropod-borne microorganism [10]. Vaccines against it are currently available and effective, yet viruses' zoonotic characteristic and occasional infections cause JEV to rank as a leading cause of high morbidity and mortality rate in Southeast Asia and the Western Pacific region [11]. 30-50% of JE patients develop permanent neuropsychiatric sequelae, while 20-30% of JE cases result in death [12]. Extensive study to develop new therapeutic strategy may be needed. This study rated inhibitory effect of ethyl acetate, methanol, and water extracts of I. indigotica, along with its related natural compounds, on JEV replication. We proved that pretreatment of I. indigotica extracts, indigo, and indirubin greatly inhibit JEV replication in vitro. These agents blocked JEV attachment, which correlated with a potent virucidal activity.

Viruses and Cells.
JEV strain T1P1 was used as previously described [13], vero cells for JEV amplification maintained in Dulbecco's modified Eagle's medium (DMEM), as well as BHK-21 cells used to determine JEV plaques grown in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS). Human promonocytic HL-CZ cells cultured in RPMI-1640 medium served to determine JEV yield in vitro.

I. indigotica Extracts and Related Marker Compounds.
Crude extract powder of I. indigotica was obtained from Sun Ten Pharmaceutical Co., Ltd., a Taiwanese manufacturer 2 Evidence-Based Complementary and Alternative Medicine of concentrated herbal extracts. For each extract tested, 1 g of powder was dissolved in 40 mL ethyl acetate or methanol, then gently shaken overnight at room temperature. Extract solutions were collected following centrifugation at 12,000 rpm for 20 min, filtered using a Whatman number 1 filter paper, then lyophilized using in a freeze dryer (IWAKI FDR-50P). Each lyophilized extract powder was kept in sterile bottles at −20 • C. Stock extract solutions (1 mg/mL) were dissolved in phosphate-buffered saline (PBS), sterilized using a 0.44 µm syringe filter and stored at −80 • C until used. Marker compounds of I. indigotica like adenosine, betulin, indigo, indirubin, tryptanthrin, lupeol, and 2benzoxazolinone were purchased from Sigma Chemical Co. (St. Louis, MO.). Stock solution of marker compounds (20 mg/mL) was dissolved in dimethyl sulfoxide (DMSO), diluted with PBS. DMSO (0.005%, 0.05%, 0.5%, and 5%) was tested as solvent control.

Cell Viability Assay.
To calculate cytotoxicity to BHK-21 cells and human promonocytic cells, cells were cultured overnight on 96-well plates. Medium containing DMSO (0.005, 0.05, 0.5, or 5%), I. indigotica extracts or marker compounds (0 µg/mL, 0.1 µg/mL, 1 µg/mL, 10 µg/mL, and 100 µg/mL) were added and incubated for another 48 hours. Living cells and total HL-CZ cell count with(out) treatment were measured by staining with 0.4% trypan blue; viability was estimated as ratio of living/total cell counts. Quadruplicate wells were analyzed for each concentration. Cytotoxic concentration showing 50% toxic effect (CC 50 ) was derived by computer program (provided by John Spouge, National Center for Biotechnology Information, National Institutes of Health).

Quantitative Assay of Virus Yields Using Plaque Assay.
To test inhibitory effect of I. indigotica on JEV yields in human promonocytic cells, HL-CZ cells infected with JEV at multiplicity of infection of 0.5 and treated with DMSO (0.005, 0.05, or 0.5%), I. indigotica extract (1, 10, and 100 µg/mL) or marker compound (0.1, 1, 10 µg/mL) at the same time. At 24 and 48 h after inoculation, cultured supernatant from (un)treated JEV-infected cells was collected for measuring virus yields by plaque assay. A 10-fold serial dilution of cultured medium was added into the well of BHK-21 cell monolayer at 37 • C for 1 h and overlaid with MEM medium containing 1.1% methylcellulose. Viral plaques were stained with naphthol blue-black dye after three-day incubation.

Plaque Reduction and Time-of-Addition Assay.
To gauge inhibitory effect of I. indigotica by time of addition on JEV replication in vitro, pretreatment (prior to infection), simultaneous treatment (at the same time as infection), and posttreatment (after entry) experiments were performed. For the pretreatment experiment, BHK-21 cell monolayer was pretreated with/without various DMSO concentrations (0, 0.005, 0.05, or 0.5%), I. indigotica extract (0, 1, 10, and 100 µg/mL) or marker compound (0, 0.1, 1, 10 µg/mL) 1-h before infection. BHK-21 cell monolayer was overlaid with MEM medium containing 1.1% methylcellulose 1 h after infection, viral plaques stained with naphthol blue-black dye after three-day incubation. For simultaneous treatment, medium with/without various DMSO concentrations (0, 0.005, 0.05, or 0.5%), I. indigotica extract (0, 1, 10, and 100 µg/mL) or marker compound (0, 0.1, 1, 10 µg/mL) was mixed along with JEV at 100 pfu, then forthwith added into the well of BHK-21 cell monolayer at 37 • C for 1 h and overlaid with MEM medium containing 1.1% methylcellulose for viral plaque assays. In posttreatment assay, BHK-21 cell monolayer was infected with JEV at 100 pfu for 1 h, followed by 1 h incubation with drug solutions and overlaid with MEM medium containing 1.1% methylcellulose, as described in plaque assay. Data represent means ± SD of three independent experiments. Inhibitory concentration showing 50% JEV plaque reduction (IC 50 ) was determined by computer program (John Spouge, National Center for Biotechnology Information, National Institutes of Health).

Mouse Protection
Assay. Groups (n = 10) of 2-weekold BALB/c mice were intracerebrally infected with 1 × 10 5 pfu of virulent JEV strain Beijing-1 then underwent three intracerebral treatments with 30 µg/100 µL of I. indigotica extract or marker compound (1 mg/kg of body weight) using 100 µL syringes at 2, 24, and 48 h after infection. Two additional groups were infected with JEV and received PBS or DMSO (0.05%) treatment as solvent controls. Survival rates were monitored every day for one week.
2.9. Statistical Analysis. ANOVA using SPSS program (version 10.1, SPSS Inc., IL, USA) or Student t-test analyzed data, P value less than 0.05 considered statistically significant.

Cytotoxicity of I. indigotica Extracts and Related Marker
Compounds. To test cytotoxicity, BHK-21 and HL-CZ cells were treated with both I. indigotica extract and related marker compounds at concentrations of 0.1-1000 µg/mL. Since diluted solutions of indigo and indirubin contained 0.0005% 0.005%, 0.05%, 0.5%, and 5% DMSO, cells were also treated with serial dilution of DMSO as solvent control.  Table 1). Other I. indigotica related marker compounds like tryptanthrin, adenosine, betulin, lupeol, and 2-benzoxazolinone showed high toxicity to both cell lines (CC 50 < 25 µg/mL). Compared to ethyl acetate and methanol extracts, indigo and indirubin manifested low toxicity to such cells, being available for in vitro and in vivo activity against JEV.    showed dose-dependent inhibition of JEV replication in HL-CZ promonocytic cells, but no time-dependent inhibitory effect on JEV production in vitro. Particularly, indigo (10 µg/mL) and indirubin (10 µg/mL) showed virus yield reduced by approximately 40% after 24 h incubation.  Table 2). Both indigo and indirubin tallied a therapeutic index (CC 50 /IC 50 ) of >10. Meanwhile, each dilution of DMSO had no significant effect on plaque reduction (data not shown).

Inhibition of JEV Replication by Pretreatment of I. indigotica Extracts
Both pre-and post-treatment plaque reduction assays tested antiviral effect of I. indigotica on JEV replication. In pretreatment assay, both indigo and indirubin pretreated before JEV adsorption showed antiviral activity similar to simultaneous treatment assay ( Figure 2 and Table 2). However, posttreatment of I. indigotica extracts, indigo and indirubin was ineffective in antiviral activity after virus entry. Results demonstrated pretreatment of I. indigotica extracts, indigo and indirubin that affects JEV replication in vitro.

Inhibition of Virus Attachment by Indigo and Indirubin.
To rate inhibitory effect of I. indigotica on virus attachment, JEV mixture (120 pfu) with I. indigotica extract, indigo, indirubin, or 0.5% DMSO (solvent control) was immediately incubated at 4 • C with BHK-21 cell monolayer to allow attachment alone. After virus attachment at 4 • C for 1 h, each mixture was removed and cell monolayer washed with PBS. Residual infectivity derived by plaque assay yielded IC 50 values of methanol extracts, indigo and indirubin below preand simultaneous treatment ( Figure 3, Table 2). However, 0.5% DMSO had no significant effect on virus attachment (data not shown). These results demonstrated a potent inhibitory effect of methanol extracts, indigo and indirubin on JEV attachment.

Virucidal Activity of Indigo and Indirubin.
To ascertain whether I. indigotica has a virucidal action by directly interfering with virus particles, JEV was preincubated with both indigo and indirubin at 4 • C for 1 h, and residual infectivity tested by plaque assay (Figure 4 and Table 2). Both indigo and indirubin exhibited concentration-dependent virucidal activity as well as significant inhibitory effect on residual infectivity compared to controls. Virucidal IC 50 values against JEV were 65.79 µg/mL of ethyl acetate extract, 22.17 µg/mL of methanol extract, 3.03 µg/mL of indigo, and 0.47 µg/mL of indirubin. Moreover, virucidal IC 50 values were below pre-, simultaneous, and posttreatment as well virus attachment assay ( Table 2), revealing that I. indigotica directly inactivated JEV particles, exhibiting a potently virucidal action.

Protection against Lethal Challenge in Mice by Indigo and Indirubin.
To investigate in vivo protective potential of I. indigotica, groups of mice were intracerebrally challenged with lethal dose of virulent JEV strain Beijing-1 and treated with extracts, indigo, indirubin, PBS, or 0.5% DMSO at 2, 24, and 48 h after infection. Survival rate of the indirubin-treated group on Day 6 after infection was 70%, starkly higher than others: for example, indigo-(50%) and ethyl acetate extract-treated (20%) ( Figure 5). None in the methanol extract-, PBS-, or DMSO-treated groups survived, indicating indirubin as superior to indigo, ethyl acetate extract better than methanol extract in mouse protection against lethal i.c. challenge with JEV.
Among related compounds, indirubin manifested potent anti-JEV activities with plaque reduction (IC 50 = 13.68 µg/mL via simultaneous treatment), virus attachment inhibition (IC 50 = 5.10 µg/mL) and virucidal inactivation (IC 50 = 0.47 µg/mL) (Figures 2-4 and Table 2). Indirubin also concentration-dependently reduced virus yield in cell cultures (Figure 1(b)). Indigo effectively inhibited JEV replication in vitro, reduced virus yield and attachment ( Figures  1(b) and 3(b)), showing greater virucidal activity (IC 50 = 3.03 µg/mL) than I. indigotica extracts. Indirubin and indigo had a potent virucidal activity through directly inactivating virus particles, linking with a better inhibition of JEV replication by pretreatment, and a significant reduction of virus attachment and yield in vitro. Similar antiviral effect of indirubin against pseudorabies virus has been also reported [5]. The inconsistency in anti-JEV abilities among reducing virus yield, virus attachment and virucidal activity could be due to the possibility that cells rapidly uptakes indirubin and indigo, then metabolizes them as inactive production, being supported in a prior report [16].
Indirubin had potent in vivo protection against intracerebral JEV challenge at lethal dose, more than indigo, ethyl acetate extract, or methanol extract ( Figure 5). Indirubin likewise regulates immunomodulatory activity on RANTES expression in influenza-infected bronchial epithelial cells [8], showing broad-spectrum antiviral activity and more effective virucidal action against JEV. In sum, I. indigotica contains potential antiviral components against JEV, and so forth through virucidal actions. Among major I. indigotica components, indirubin manifests potential for antiviral activity against JEV infection, which could yield new anti-JEV agents.