Excoecarianin, Isolated from Phyllanthus urinaria Linnea, Inhibits Herpes Simplex Virus Type 2 Infection through Inactivation of Viral Particles

Abstract

Phyllanthus urinaria Linnea (Euphorbiaceae) is one of the traditional medicinal plants widely used by oriental people to treat various diseases. We have previously demonstrated that the acetone extract of P. urinaria inhibits herpes simplex virus type 2 (HSV-2) but not HSV-1 infection. In a continuing effort to clarify the antiviral mechanisms of P. urinaria, we isolated the pure compound excoecarianin from the whole plant of P. urinaria through acetone extraction, and investigated its anti-HSV-1 and HSV-2 activities. Our results indicated that excoecarianin protected Vero cells from HSV-2 but not HSV-1 infection, and its 50% inhibitory concentration (IC(50)) was 1.4 ± 0.1 μM. The antiviral effective concentration of excoecarianin did not affect the viability or the morphology of Vero cells. Although excoecarianin inhibited HSV-2 infection, the inhibitory effect, however, was most prominent when excoecarianin was concurrently added with the virus. Pretreatment of Vero cells with excoecarianin with removal of the drug prior to infection did not yield any antiviral effects, and the same observation was made for post viral entry treatment. Subsequent studies revealed that excoecarianin inactivated HSV-2 virus particles to prevent viral infection. A synergistic antiviral effect against HSV-2 was also observed when Vero cells were treated with a combination of acyclovir (ACV) and excoecarianin. These results suggested that excoecarianin merits to be further explored as an entry inhibitor against HSV-2 and could potentially be investigated for combinatorial drug treatment with nucleoside analogues such as ACV in therapeutic management of HSV-2 infection.

Figure 1

Chemical structure of excoecarianin.

Figure 2

The cytotoxic effect of excoecarianin (open bars) and ACV (dotted bars) toward Vero cells as determined by XTT assay. Various concentrations of excoecarianin or ACV were added to Vero cells. After 72 h of incubation, the XTT solution was added and then the optical densities were measured. The cytotoxic effect of excoecarianin and ACV were evaluated and the 50% cytotoxic concentration (CC₅₀) was calculated. Each bar represents the mean ± SD of three independent experiments. The asterisk indicates significant difference between test sample and solvent control (P < .05).

Figure 3

The morphology of Vero cells without treatment (a) and with treatment of ACV (b) or excoecarianin (c)-(d). Vero cells were seeded onto 24-well culture plates at density of 1 × 10³ cells per well. After 4 h, excoecarianin or ACV was added. The cells were incubated for 7 days, and the cellular morphology was examined under phase-contrast microscope.

Figure 4

Inhibitory effect of excoecarianin (open bars) and ACV (dotted bars) against HSV-2 infection in Vero cell as determined by plaque reduction assay. Vero cells were incubated with 100 pfu of HSV-2 and different concentrations of excoecarianin or ACV. After 1 h, an overlay medium containing 1% methylcellulose was added. On Day 3 post-infection, the cell monolayer was stained with crystal violet and the virus plaques formed were counted. The percentage of inhibition was calculated by comparing the plaque number of compound-treated group to that of the untreated group. The concentrations of excoecarianin and ACV that inhibited 50% of HSV-2 infection (IC₅₀) were determined. Each bar represents the mean ± SD of three independent experiments. The asterisk indicates significant difference between test sample and solvent control (P < .05).

Figure 5

Synergistic antiviral activity of ACV and excoecarianin in Vero cells. IC₅₀ values were derived from the data shown in Table 3 and used to construct the isobologram. FIC₅₀ of ACV represents the ratio of the IC₅₀ of ACV in the presence of a constant concentration of excoecarianin to the IC₅₀ of ACV alone. The x-axis represents the ratio of the fixed concentration of excoecarianin to the IC₅₀ of excoecarianin alone. In this representation, displacement of the experimental data points to the left of the theoretical line is indicative of synergistic behavior.

Figure 6

The time-of-addition effect of excoecarianin against HSV-2 infection. Excoecarianin added prior to virus infection and then washed out did not protect the cells from HSV-2 infection. The addition of excoecarianin after 2 h of the virus inoculation also did not inhibit HSV-2 infection. The compound can inhibit HSV-2 infection only when it was added concurrently with the virus infection.