The cytotoxicity of the honeysuckle extracts in MDCK and Hela cells was examined. As shown in Fig. 1A, upon treatments with the four honeysuckle extracts at 25 μg/mL, cell viability remained at least 95% at 48 h post-infection, indicating that the treatments induced limited cytotoxicity at concentrations less of 25 μg/mL. In addition, the CC50 and EC50 values of the four honeysuckle extracts on the influenza viruses were determined (Table 1). The total extract induced the lowest cytotoxicity, with a CC50 value of 350.0 μg/mL in MDCK cells. The acids-flavonoids mixture showed the most effective antiviral activity against H1N1 and H3N2 with EC50 values of 3.8 μg/mL and 4.1 μg/mL, respectively.
Figure 1. Inhibitory effects of honeysuckle extracts on influenza viruses H1N1 and H3N2 in MDCK cells. A Cellular toxicity of honeysuckle extracts in MDCK cells. B Microscopic images for H1N1 virusinfected, 20 μg/mL honeysuckle extracts-treated, 2 μg/mL OCtreated, and 2 μg/mL ribavirin-treated MDCK cells at 48 h post-infection. C Inhibitory rates of honeysuckle extracts (1-20 μg/mL) against influenza virus A/FM/1/47 (H1N1), and D H3N2 in MDCK cells. 2 μg/mL oseltamivir carboxylate and ribavirin were used as positive controls. Inhibitory rates of honeysuckle extracts (20 μg/mL) against H1N1 (E) and H3N2 (F) influenza viruses with different infection protocols in MDCK cells. The results were presented as the mean ± S.D. of three independent experiments, *P < 0.05; **P < 0.01; ***P < 0.001, compared with the oseltamivir-treated group, #P < 0.05; ##P < 0.01; ###P < 0.001, compared with the ribavirin-treated group.
Compounds CC50a H1N1 H3N2 MDCK HeLa EC50b SIc EC50 SI Acids extract 139.8 ± 9.3 143.3 ± 6.0 15.6 ± 1.5 8.9 5.6 ± 0.6 24.9 Flavonoids extract 82.4 ± 0.8 76.9 ± 1.4 17.2 ± 3.8 4.8 14.1 ± 2.5 5.9 Total extract 350.0 ± 14.5 178.1 ± 2.7 14.3 ± 7.0 25.2 7.4 ± 1.0 48.6 Acids-flavonoids 88.3 ± 3.9 116.5 ± 2.9 3.8 ± 0.3 23.5 4.1 ± 0.5 21.8 Oseltamivir >500 2.6 ± 0.1 >192 3.1 ± 0.1 >161 Ribavirin 13.7 3.5 ± 0.1 3.9 NDd ND The results were the mean ± S.D.of three independent experiments.
aConcentration of drugs required to reduce the viability of normal MDCK cells by 50% (μg/mL).
bConcentration of drugs required to improve the viability of influenza virus-infected cells by 50% (μg/mL).
cSelectivity index, CC50/EC50.
Table 1. Cytotoxicity and antiviral activity of honeysuckle extracts.
To investigate whether these honeysuckle extracts can inhibit the replication of the H1N1 and H3N2 influenza viruses, a CPE reduction assay was performed in MDCK cells. The CPE reduction was confirmed by direct microscopic observation. Each honeysuckle treatment cell group had a considerably lower CPE than the placebo group (H1N1 infected cells treated with DMSO) (Fig. 1B). All four honeysuckle treatment protected MDCK cells from influenza virus infection in a dosedependent manner while inducing low cytotoxicity (Fig. 1C, 1D). In addition, acids-flavonoids mixture exhibited stronger suppressive effects than the three extracts in MDCK cells, especially at a final concentration of 20 μg/mL, with inhibition rates of 80.4% and 81.4% against H1N1 and H3N2, respectively. At a concentration of 2.5 μg/mL, the acids-flavonoids mixture had an anti-influenza-virus activity effect against H1N1 and H3N2 similar to that of oseltamivir and ribavirin.
To clarify the possible anti-influenza mechanism of the four honeysuckle extracts treatments, their inhibitory effects on influenza virus replication were examined using three different infection protocols. As shown in Fig. 1E-1F, when 20 μg/mL honeysuckle extracts were added 2 h post-infection, the inhibition rates against H1N1 and H3N2 viruses were much higher than those observed after the 'pre-infection' or 'co-infection' treatment of infected cells, indicating that honeysuckle treatments mainly inhibited the replication and release of influenza virus but not viral adsorption or penetration.
Taken together, these results indicate that all four honeysuckle extracts treatments effectively inhibited the replication of H1N1 and H3N2 influenza viruses in MDCK cells, and the honeysuckle acids-flavonoids mixture had the strongest inhibitory effects in vitro.
In addition to wild type influenza viruses, we also investigated the inhibitory effects of the honeysuckle extracts treatments on the oseltamivir-resistant mutant strain H1N1-H275Y in vitro. As shown in Fig. 2, four honeysuckle extracts obviously inhibited the replication of the influenza virus H1N1-H275Y in a dose-dependent manner in MDCK cells. The inhibition rate of the acids-flavonoids mixture against H1N1-H275Y virus was 47.8% at a concentration of 20 μg/mL, while the inhibition rates of oseltamivir and ribavirin were only 3.5% and 14.8%, respectively, showing that acids-flavonoids mixture can effectively inhibit the replication of the oseltamivir-resistant virus in vitro.
Figure 2. Inhibitory effects of honeysuckle extracts on oseltamivir-resistant mutant influenza virus H1N1-H275Y in MDCK cells. 2 μg/mL oseltamivir carboxylate and ribavirin were used as positive controls. The results were presented as the mean ± S.D. of three independent experiments, *P < 0.05; **P < 0.01; ***P < 0.001, compared with the oseltamivir-treated group, #P < 0.05; ##P < 0.01; ###P < 0.001, compared with the ribavirin-treated group.
The inhibitory efficacy of four honeysuckle extracts on influenza A virus in vivo was evaluated in an H1N1-infected mouse model. The experimental protocol is shown in Fig. 3A. On 'day 0' the mice were infected intranasally with a lethal dose of H1N1 virus. At 2 h post infection, drugs were delivered orally to mice once daily for 5 days (days 0-4).
Figure 3. Therapeutic efficacy of honeysuckle extracts against the influenza virus in mice. A Experimental protocol of testing honeysuckle extracts in H1N1-infected mice. Fourteen uninfected ICR mice were as control group. ICR mice were intranasally infected with a mouse-adapted influenza virus H1N1 (8 × LD50). At 2 h post-infection, fourteen mice per group were treated with saline (placebo group), four honeysuckle extracts (600 mg/kg/d), or ribavirin (100 mg/kg/d) twice daily for 5 successive days (days 0-4) by oral gavage, respectively. On day 5 and day 6 post-infection, four mice per group were euthanized for lung pathological examinations. Survival rate (B) and body weight (C) were monitored for 14 days (n = 10). Viral load (D), Lung index (E) and Lung score (F) of mice infected with influenza virus H1N1 (n = 4). G H & E staining of sectioned lungs (n = 4). The histopathological changes are indicated by arrows. Data shown as the mean ± S.D. *P < 0.05; **P < 0.01; ***P < 0.001, compared with the placebo group.
As shown in Fig. 3B, acids extract-treated mice were obviously protected from death caused by influenza virus, whereas all the mice administered the placebo died within 8 days. Treatment of the mice with 600 mg/kg/d of honeysuckle acids extract, flavonoids extract, total extract or acids-flavonoids mixture increased survival to 30%, 10%, 10% and 20%, respectively. In addition, administration of the four honeysuckle extracts effectively protected the infected mice from body weight loss (Fig. 3C). Despite a similar trend in body weight loss during the first 9 days post-infection, the mice administered the honeysuckle extracts treatments regained weight starting on day 10, whereas infected mice treated with placebo showed significant body weight loss until their death.
On 'day 5' and 'day 6' we used qPCR to detect the viral load in the lungs of the treated mice. Acids extract and ribavirin treatments significantly inhibited viral RNA expression on day 5 but only slightly suppressed viral RNA expression on day 6 (Fig. 3D). Four mice per group were randomly selected and euthanized for use in lung pathological examinations on day 5. The four honeysuckle extracts treatments effectively suppressed the influenzainduced increases in lung indexes to a different degree (Fig. 3E). At a dosage of 600 mg/kg/d, the lung index of the honeysuckle acids extract-treated mice was 1.4, a statistically significant decrease compared to that of the placebo group (lung index: 1.9). The four honeysuckle extracts treatments administered at a dosage of 600 mg/kg/d protected the lungs of mice from damage caused by influenza virus infection. On day 5, features of extensive lung damage, such as alveolar wall congestion, infiltration of inflammatory cells and bronchial epithelium necrosis, were observed in the placebo group (lung score: 7.63) (Fig. 3F, 3G). In the honeysuckle acids extract-treated mice, the lung damage was notably attenuated (lung score: 5.25).
These data indicated that the honeysuckle acids extract significantly reduced body weight loss, inhibited lung damage and prevented the death of influenza-infected mice. The anti-influenza virus effects of the honeysuckle acids extract was slightly weaker than that of the acidsflavonoids mixture in vitro, but in vivo, and the pharmaco dynamic study demonstrated the potent therapeutic efficacy of the honeysuckle acids extract against infection with influenza virus H1N1.
The results of the time-of-drug addition experiment indicated that honeysuckle extracts primarily inhibited the replication and release of the influenza virus after entrance into host cells (Fig. 1E, 1E). Influenza virus neuraminidase can hydrolyze sialic acid residues in progeny virus to promote the release of progeny virus particles from host cells, thus playing an important role in the spread of the influenza virus. Therefore, we speculated that honeysuckle extracts may inhibit the neuraminidase activity of influenza viruses. The inhibitory effects of the honeysuckle extracts treatments on the neuraminidase activity of H1N1, H1N1-H275Y, H3N2 influenza viruses, CHO-expressed H5N1-NA, CHO-expressed H7N9-NA and CHO-expressed H7N9-R294K-NA were examined using MUNANA, a specific fluorescent substrate. Figure 4 shows that the four honeysuckle extracts treatments significantly inhibited the neuraminidase activity of multi-influenza viruses in a dose-dependent manner. Among the four honeysuckle extracts treatments, the flavonoids extract showed the best antineuraminidase activity. Moreover, the four honeysuckle extracts treatments had a greater inhibitory effect against recombinant NA from the H7N9 influenza virus than against NA from the other influenza viruses. As shown in Table 2, the IC50 value of the honeysuckle flavonoids extract against NA of H7N9 influenza virus was 24.7 μg/mL. These results demonstrated that the honeysuckle extracts treatments had broad-spectrum inhibitory activity against NA in multiple influenza viruses, including the oseltamivir-resistant mutant strains.
NA IC50a ± SD (μg/mL) Acids extract Flavonoids extract Total extract Acids-flavonoids H1N1 112.3 ± 17.7 90.9 ± 8.6 305.1 ± 23.7 100.1 ± 11.4 H3N2 332.6 ± 34.5 196.0 ± 23.4 947.3 ± 39.5 203.8 ± 9.9 H5N1 78.8 ± 5.5 54.7 ± 3.4 283.9 ± 29.0 63.8 ± 0.6 H7N9 55.9 ± 5.1 24.7 ± 2.3 170.0 ± 5.2 35.2 ± 3.1 H1N1-H275Y 150.4 ± 13.6 108.4 ± 17.0 350.1 ± 36.9 125.7 ± 14.7 H7N9-R294K 80.1 ± 6.3 44.1 ± 5.4 291.2 ± 26.2 53.1 ± 6.0 The results were the mean ± S.D. of three independent experiments.
aConcentration of drugs required to reduce the activity of neuraminidase by 50%.
Table 2. IC50 of honeysuckle extracts against neuraminidase activity of influenza A virus.