Water extract of Pueraria lobata Ohwi has anti-viral activity against human respiratory syncytial virus in human respiratory tract cell lines

Abstract

Human respiratory syncytial virus (HRSV) infects all age groups and causes bronchiolitis, pneumonia, and acute respiratory distress syndrome with a significant mortality rate. To date, only ribavirin has been used to manage HRSV infection. However, ribavirin is expensive with an only modest effect. Furthermore, ribavirin has several side effects, which means it has limited clinical benefit. Pueraria lobata Ohwi (P. lobata) is a common ingredient of Ge-Gen-Tang (Kakkon-to) and Sheng-Ma-Ge-Gen-Tang (Shoma-kakkon-to), which are prescriptions of Chinese traditional medicine proven to have antiviral activity against HRSV. Therefore, it was hypothesized that P. lobata might be effective against HRSV. To find a cost-effective therapeutic modality, both human upper (HEp-2) and lower (A549) respiratory tract cell lines were used to test the hypothesis that P. lobata could inhibit HRSV-induced plaque formation. Results showed that the water extract of P. lobata was effective (p < 0.0001) against HRSV-induced plaque formation. P. lobata was more effective when given prior to viral inoculation (p < 0.0001) by inhibiting viral attachment (p < 0.0001) and penetration (p < 0.0001). However, supplementation with P. lobata could not stimulate interferon secretion after HRSV infection. In conclusion, P. lobata has antiviral activity against HRSV-induced plaque formation in airway mucosa mainly by inhibiting viral attachment and internalization. Further identification of effective constituents could contribute to the prevention of HRSV infection.

Keywords: Chemoprevention; Ge-Gen-Tang; Human respiratory syncytial virus; Pueraria lobata; Respiratory tract infection.

Figure 1

Cytotoxicity assay. Pueraria lobata did not show any cytotoxicity against host cells up to the concentration of 3000 μg/mL after 3 days' incubation. Data are presented as mean ± standard deviation of three triplicates.

Figure 2

Antiviral effect assay. Both Pueraria lobata (A) and ribavirin (B) were dose‐dependently (p < 0.0001) effective against human respiratory syncytial virus as determined by plaque reduction assay after 3 days' incubation. P. lobata was more effective on A549 cells than on HEp‐2 cells (p < 0.0001). By contrast, ribavirin did not show this difference. Data are presented as mean ± standard deviation of three triplicates. *p < 0.05; **p < 0.001; and ***p < 0.0001 were compared to the viral control (0 μg/mL). **** p < 0.05 was compared between the different cell types at the same concentrations.

Figure 3

Time of addition assay. The effect of Pueraria lobata was dose‐dependent (p < 0.0001) against human respiratory syncytial virus on HEp‐2 (A) and A549 cells (B). This activity was also affected by the time of addition. P. lobata was more effective when given prior to viral inoculation (p < 0.0001). Data are presented as mean ± standard deviation of three triplicates. *p < 0.05; **p < 0.001; and ***p < 0.0001 were compared to the viral control (0 μg/mL).

Figure 4

Attachment assay. of Pueraria lobata was dose‐dependently effective against viral attachment in HEp2 cells and A549 cells (p < 0.0001), particularly on A549 cells (p < 0.0001). Data are presented as mean ± standard deviation of two triplicates. *p < 0.05; **p < 0.001; and ***p < 0.0001 were compared to the control group (0 μg/mL).

Figure 5

Internalization assay. Pueraria lobata was time‐dependently (p < 0.0001) and dose‐dependently (p < 0.0001) effective against viral penetration in HEp‐2 cells (A) and A549 cells (B). Data are presented as mean ± standard deviation of three triplicates. *p < 0.05; **p < 0.001; and ***p < 0.0001 were compared to the viral control (0 μg/mL).

Figure 6

Interferon (IFN)‐β assay. Human respiratory syncytial virus infection increased IFN‐β secretion in HEp‐2 (A) and A549 (B) cells. P. lobata stimulated HEp‐2 (A) and A549 (B) cells to secrete IFN‐β only at high concentrations without human respiratory syncytial virus infection. Data are presented as mean ± standard deviation of three triplicates. *p < 0.05; **p < 0.001; and ***p < 0.0001 were compared to the group (0 μg/mL). **** p < 0.05 was compared to the cell control (0 μg/mL without viral inoculation).