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

Abstract

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.

Figure 1

Inhibition of virus yield by I. indigotica extract, indigo and indirubin. HL-CZ cells were infected with JEV at a MOI of 0.5 at the same time as treatment with serial dilution of I. indigotica extracts (a) indigo and indirubin (b). Cultured supernatants in infected cells were harvested at 24 and 48 h after infection. Virus yield was performed as described in plaque assay.

Figure 2

Plaque reduction of JEV by I. indigotica and its major components in pre-, simultaneous, and post-treatment assays. Serial dilutions of ethyl acetate and methanol extracts of I. indigotica (a) and its components indigo and indirubin (b) were pretreated before virus absorption, simultaneously with virus infection, or posttreated after virus absorption (JEV at 100 pfu/well). BHK-21 cell monolayer was incubated with virus/compound mixture at 37°C for 1 h, then overlaid with MEM medium containing 1.1% methylcellulose. Viral plaques were stained with naphthol blue-black dye after 3 days of incubation.

Figure 3

Inhibition of virus attachment by I. indigotica extract, indigo and indirubin. I. indigotica extracts (a) indigo and indirubin (b) were serially diluted and mixed with JEV (120 pfu), each mixture incubated 1 h with BHK-21 cell monolayers at 4°C. Then virus/compound mixture was removed and cell monolayer washed with cold PBS. Residual infectivity was performed as described in plaque assay.

Figure 4

Virucidal activities of I. indigotica and its major components. I. indigotica extracts (a) indigo and indirubin (b) were serially diluted and mixed with JEV. Each virus/compound mixture was incubated at 4°C for 1 h, then added onto the BHK-21 cell monolayer at 37°C for another 1 h. The virus/compound mixture was removed from 6-well plates and cell monolayer washed with PBS. Residual infectivity was performed as described in plaque assay.

Figure 5

Mouse protection against lethal JEV challenge by I. indigotica. Groups of 2-week-old BALB/c mice were intracerebrally infected with JEV at 1 × 10⁵ pfu, then underwent three intracerebral treatments with I. indigotica extract, indigo, or indirubin (1 mg/kg of body weight) 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 daily.