Antiviral Activity of Total Flavonoid Extracts from Selaginella moellendorffii Hieron against Coxsackie Virus B3 In Vitro and In Vivo

Abstract

The antiviral activity of total flavonoid extracts from Selaginella moellendorffii Hieron and its main constituents amentoflavone were investigated against coxsackie virus B3 (CVB3). When added during or after viral infection, the extracts and amentoflavone prevented the cytopathic effect (CPE) of CVB3, as demonstrated in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay, with a 50% inhibitory concentration (IC50) from 19 ± 1.6 to 41 ± 1.2 μ g/mL and 25 ± 1.2 to 52 ± 0.8 μ g/mL, respectively. KM mice were used as animal models to test the extracts' activity in vivo. Oral administration of the total flavonoid extracts at 300 mg/kg/day significantly reduced mean viral titers in the heart and kidneys as well as mortality after infection for 15 days. The experimental results demonstrate that in vitro and in vivo the model mice infected with CVB3 can be effectively treated by the total flavonoid extracts from Selaginella moellendorffii Hieron.

Figure 1

Morphology changes of HEp-2 cells infected with CVB3. (a) Normal control demonstrated that these HEp-2 cells were typical with normal morphology. (b) Viral control demonstrated that most of the HEp-2 cells lost their normal morphology and appeared to be round in shape. Most of them were detached. (c) TFE group demonstrated that some HEp-2 cells had normal morphology and quantity.

Figure 2

Direct virucidal activity of EAE, TFE, and amentoflavone. The average viral inhibition rate of samples (%) was detected by MTT assay, when they were treated during infection. Viral suspensions were cocultured with the dilutions of the samples (50–6.25 μg/mL) at 37°C for 1 h in a 5% CO₂ atmosphere.

Figure 3

Virucidal activity of EAE, TFE, and amentoflavone after the CVB3 infection. The average viral inhibition rate of samples (%) was detected by MTT assay. The HEp-2 cells were infected with viruses for 1 h at 37°C in a 5% CO₂ atmosphere. Then cells were washed with PBS and added with different doses of the samples (50–6.25 μg/mL).

Figure 4

The body weight of CBV3-infected mice during the 15-day period. KM mice were infected intraperitoneally with CVB3 (10⁵TCID₅₀/0.2 mL). 24 h after infection, mice were treated by oral gavage with 300 mg/kg/day TFE for 15 days. The virus control group and the normal control group received water instead of the chemicals. Body weight was recorded daily.

Figure 5

Pathologic features of hearts and kidneys (HE staining, ×200). (a) Normal control of heart tissue; (b) infected control of heart tissue; (c) TFE-treated group of heart tissue; (d) normal control of kidney tissue; (e) infected control of kidney tissue; (f) TFE-treated group of kidney tissue. Animals of each group were sacrificed on day 15 after viral exposure for pathological examination.