Anti-Influenza Activity of Betulinic Acid from Zizyphus jujuba on Influenza A/PR/8 Virus

Abstract

Betulinic acid, a pentacyclic triterpene isolated from Jujube tree (Zizyphus jujuba Mill), has been known for a wide range of biological and medicinal properties such as antibacterial, antimalarial, anti-inflammatory, antihelmintic, antinociceptive, and anticancer activities. In the study, we investigated the antiviral activity on influenza A/PR/8 virus infected A549 human lung adenocarcinoma epithelial cell line and C57BL/6 mice. Betulinic acid showed the anti-influenza viral activity at a concentration of 50 μM without a significant cytotoxicity in influenza A/PR/8 virus infected A549 cells. Also, betulinic acid significantly attenuated pulmonary pathology including increased necrosis, numbers of inflammatory cells and pulmonary edema induced by influenza A/PR/8 virus infection compared with vehicle- or oseltamivir-treated mice in vivo model. The down-regulation of IFN-γ level, which is critical for innate and adaptive immunity in viral infection, after treating of betulinic acid in mouse lung. Based on the obtained results, it is suggested that betulinic acid can be the potential therapeutic agent for virus infection via anti-inflammatory activity.

Keywords: A549; Betulinic acid; Inflammation; Influenza A/PR/8; Zizyphus jujuba.

Fig. 1.

Antiviral activity BeA against influenza A/PR/8 virus in A549 cells. Antiviral activity of BeA was assessed against influenza A/PR/8 virus in A549 cells. The indicated concentrations of BeA was determined using a concentration series of 0.4, 2, 10, and 50 μM. After 48 h of incubation, the antiviral activity was investigated by CPE reduction assay using SRB. (A) The structure of betulinic acid. (B) Absorbance in each well was measured using microplate reader. ^***p<0.0001 and n.s. for not significant. (C) The percent value was calculated by absorbance value as mentioned in Materials and Methods. Results are presented as the mean ± SD of the absorbance and percentage values obtained from three independent experiments carried out in triplicate. ^*p<0.001; ^**p<0.03; ^***p<0.0001 and n.s. for not significant.

Fig. 2.

Active concentration of BeA was determined. (A) 5×10³ pfu/30 μl of influenza A/PR/8 virus was inoculated. The indicated treatment was started after infection. BeA and oseltamivir were orally administerd daily for 7 days and the survival rate of mice were measured. The body weights of mice were measured. (B) Influenza A/PR/8 virus gene expressions in the lung tissue were detected by real-time PCR 7 days after influenza A/PR/8 virus infection. ^**p<0.05 and n.s. for not significant.

Fig. 3.

Anti-influenza virus activity of BeA and oseltamivir in the mice. Mice were infected with 5×10³ pfu/30 μl of influenza A/PR/8 virus, orally administered with BeA and oseltamivir for 7 days after virus infection. Mice were sacrificed at 7 days post infection, and lung sections were prepared as described in Materials and Methods. (A) Representative H&E stained samples of lung section from uninfected or influenza A/PR/8 virus-infected mice were shown at 200× magnification. Infected mice were treated with BeA or oseltamivir. (B) Pathological grade of mice which were administrated orally for 7 days after infection. ^**p<0.01; ^***p<0.001, and n.s. for not significant.

Fig. 4.

Cytokine quantitation changed from BeA or oseltamivir in mice. Lung inflammation cytokine levels were evaluated according to ELISA. C57BL/6 mice were intranasally infected with 5×10³ pfu/30 μl of influenza A/PR/8 virus, and orally administered with BeA (10 mg/kg) and oseltamivir (5 mg/kg). Orally administration daily for 7 days and sacrificed in mice after 6 hrs. Proinflammatory cytokines and chemokine were measured in lung by ELISA. ^*p<0.02 and n.s. for not significant.