ANTI-INFLUENZA VIRUS COMPOSITION CONTAINING PONCIRUS TRIFOLIATA EXTRACT AS ACTIVE INGREDIENT

Abstract

The present invention relates to an anti-influenza virus composition containing a Poncirus trifoliata extract as an active ingredient. The composition of the present invention inhibits influenza virus replication and infection, thereby exhibiting excellent effects in the prevention and treatment of influenza virus infection. The present invention provides a composition for preventing, treating, or alleviating diseases caused by influenza virus.

Claims

1. An anti-influenza virus composition containing a Poncirus trifoliata extract as an active ingredient.

2. The anti-influenza virus composition of claim 1, wherein the Poncirus trifoliata extract is a Poncirus trifoliata seed extract.

3. The anti-influenza virus composition of claim 1, wherein the influenza virus is influenza A virus, influenza B virus, or influenza C virus.

4. The anti-influenza virus composition of claim 1, wherein the influenza A virus is H1N1, H3N2, H5N1, or H7N9.

5. The anti-influenza virus composition of claim 1, wherein the composition is a pharmaceutical composition for preventing or treating diseases caused by influenza viruses.

6. The anti-influenza virus composition of claim 1, wherein the composition is a functional food composition for preventing or treating diseases caused by influenza viruses.

7. The anti-influenza virus composition of claim 1, wherein the composition is an animal feed additive composition for preventing or treating diseases caused by influenza viruses.

8. The anti-influenza virus composition of claim 1, wherein the composition is a disinfectant composition for preventing diseases caused by influenza viruses.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0057] FIG. 1a is a graph showing an inhibitory effect of a Poncirus trifoliata seed extract on the binding of influenza A virus H1N1 to MDCK cells. FIG. 1b is a graph showing cell viability of MDCK cells according to the concentration when MDCK cells were treated with Poncirus trifoliata seed extract or Tamiflu at indicated concentrations and then infected with influenza A virus H1N1. Attached are images of the cells treated with Poncirus trifoliata seed extract or Tamiflu at the indicated concentrations in the test.

[0058] FIGS. 2a to 2d are graphs showing cell viability of MDCK cells according to the concentration when MDCK cells infected with influenza A virus H1N1(a), H5N1(b), H3N2(c), or influenza B virus (d) were treated with Poncirus trifoliata seed extract or Tamiflu at the indicated concentrations. Attached are images of the cells infected with influenza A virus H1N1 and then treated with Poncirus trifoliata seed extract or Tamiflu at the indicated concentrations in the test.

MODE FOR CARRYING OUT THE INVENTION

[0059] Hereinafter, the present invention will be described in detail with reference to examples. These examples are only for illustrating the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

EXAMPLES

Materials and Methods

[0060] Preparation of Poncirus trifoliata Seed Extract

[0061] 100 g of pulverized dried Poncirus trifoliata seeds were extracted in 1 L of ethanol for 2 days, followed by filtration, and then the collected Poncirus trifoliata seed ethanol extract was vaporized under reduced pressure for 2-3 days, followed by freeze-drying, to give an extract.

Cell Culture

[0062] The MDCK cell line used in the test was incubated in conditions of 37° C. and 5% CO.sub.2. The cells were maintained in an Eagle's minimum essential medium (EMEM, Gibco) containing 10% fetal bovine serum (FBS, Hyclone Thermo Scientific) and 1% penicillin-streptomycin solution (Gibco).

Investigation on Cell Binding of Influenza Virus

[0063] MDCK cells dispensed in the 96-well plate were cultured at 4° C. for 1 hour. The cell line was infected with a mixture of 100 TCID.sub.50 of influenza virus H1N1 and different concentrations of Poncirus trifoliata seed extract or Tamiflu, cultured at 4° C. for 3 hours, washed twice with PBS, and again incubated at 37° C. After 48 hours, cell viability was measured. The cell viability was directly observed under a microscope, and the cell viability was again quantified and digitized using MTT assay kit (Dae ill lab servers co. Seoul. Korea). EC.sub.50 value is a concentration of an antiviral drug at a section in which cell viability is 50%.

Investigation on Influenza Virus Infection Preventive Effect of Poncirus trifoliata Seed Extract

[0064] 1×10.sup.4 MDCK cells were dispensed in each well of the 96-well plate, incubated at 37° C. for 16 hours, and washed twice with PBS, and then the MDCK cells were treated with different concentrations of Poncirus trifoliata seed extract or Tamiflu. The cells were again incubated at 37° C. for 6 hours, and washed twice with PBS, and then the cells were infected with 100 TCID.sub.50 of influenza virus H1N1, followed by incubation at 37° C. After two hours, the cells were washed three times with PBS to remove uninfected virus. Then, the cells were put in a virus growth medium (EMEM, 0.3% BSA, 1% P/S, 0.0005% tyrosine), followed by incubation at 37° C. for 48 hours, and then the cytopathic effect (CPE) was observed.

Investigation on Antiviral Activity of Poncirus trifoliata Seed Extract

[0065] 1×10.sup.4 MDCK cells were dispensed in each well of the 96-well plate, incubated at 37° C. for 16 hours, and washed twice with PBS, and then the MDCK cells were infected with 100 TCID.sub.H of influenza A virus H5N1, H1N1, or H3N2 or influenza B virus, respectively, at 37° C. for 2 hours, followed by washing twice with PBS. The cells infected with the influenza virus were treated with different concentrations of Poncirus trifoliata seed extract or Tamiflu, and then incubated at 37° C. for 48 hours. The antiviral effect of the Poncirus trifoliata seed extract was expressed by the cytopathic effect (CPE) inhibitory effective concentration 50% (EC.sub.50) value. The cytotoxic concentration 50% value (CC.sub.50) of the Poncirus trifoliata seed extract was determined based on the cellular morphological transformation. The anti-influenza virus capacity of the Poncirus trifoliata seed extract was expressed as selectivity index (SI), which is the CC.sub.50 value divided by the EC.sub.50 value.

Results

[0066] Virus Binding Inhibitory Effect of Poncirus trifoliata Seed Extract

[0067] A cell line was infected with a mixture of influenza virus H1N1 and different concentrations of Poncirus trifoliata seed extract or Tamiflu, followed by incubation. As a result, Tamiflu showed no antiviral effect since all the cells were infected with the virus, while the Poncirus trifoliata seed extract showed an EC.sub.50 value of 0.25 μg/ml, indicating an effect of inhibiting the binding of the virus to cells (FIG. 1a).

Virus Preventive Effect of Poncirus trifoliata Seed Extract

[0068] The MDCK cells cultured by the test method above were treated with different concentrations of Poncirus trifoliata seed extract or Tamiflu, and then the cells were infected with influenza virus H1N1, followed by incubation at 37° C. for 48 hours, and the CPE of cells was observed. As a result, all the cells treated with Tamiflu were dead due to infection with the virus, while the EC.sub.H value was 123.5 μg/ml for the treatment with Poncirus trifoliata seed extract (FIG. 1b). In addition, as a result of observing the cells using a microscope, the treatment with Tamiflu showed an infected cell state, while the treatment with 123.5 μg/ml Poncirus trifoliata seed extract showed that the cells were not infected with the virus, similar to normal cells (FIG. 1b).

[0069] These results show that the Poncirus trifoliata seed extract, unlike Tamiflu, inhibited the cell infection by viruses, suggesting the possibility that the Poncirus trifoliata seed extract has a high preventive effect against influenza.

Antiviral Activity of Poncirus trifoliata Seed Extract

[0070] MDCK cells were infected with influenza A virus H1N1, H3N2, or H5N1, or influenza B virus by the test method above, and then treated with different concentrations of Poncirus trifoliata seed extract or Tamiflu, to analyze antiviral activity (post-treatment assay). The antiviral effect of the Poncirus trifoliata seed extract exhibited the CPE inhibitory effective concentration 50% value (EC.sub.50) by viral infection, and the cytotoxic concentration 50% value (CC.sub.50) of the Poncirus trifoliata seed extract was determined based on the cellular morphological transformation. The EC.sub.50 value and CC.sub.50 value were calculated by Reed and Muench methods. The anti-influenza virus capacity of the Poncirus trifoliata seed extract was expressed as selectivity index (SI), which is the CC.sub.50 value divided by the EC.sub.50 value. Table 1 shows comparative results of anti-influenza viral activity of Tamiflu and the Poncirus trifoliata seed extract on influenza A virus H1N1, H3N2, or H5N1, or influenza B virus in MDCK cells.

TABLE-US-00001 TABLE 1 CC.sub.50 EC.sub.50 Virus strain Compound (μg/ml) (μg/ml) SI H1N1 Poncirus trifoliata seed 3333.3 0.07 4761 extract H1N1 Tamiflu 1111.1 3.5 317 H3N2 Poncirus trifoliata seed 3333.3 1.5 2222 extract H3N2 Tamiflu 1111.1 3.5 317 H5N1 Poncirus trifoliata seed 3333.3 0.5 6666 extract H5N1 Tamiflu 1111.1 1.5 740 Influenza B Poncirus trifoliata seed 3333.3 13.7 243 virus extract Influenza B Tamiflu 1111.1 13.7 81 virus

[0071] As can be seen from table 1, as for Tamiflu used as a positive control, the CC.sub.50 value was 1111.1 μg/ml, and the EC.sub.50 value was 3.5 μg/ml for H1N1 and H3N2, 1.5 μg/ml for H5N1, and 13.7 μg/ml for the influenza B virus. As for the Poncirus trifoliata seed extract, the cytotoxicity was not shown at 3333.3 μg/ml, and the EC.sub.50 value was 0.07 μg/ml for H1N1, 1.5 μg/ml for H3N2, 0.5 μg/ml for H5N1, and 13.7 μg/ml for influenza B virus.

[0072] In the results with respect to H1N1 infected strain, as for Tamiflu, the EC.sub.50 value showing an anti-viral effect of 50% or more was observed at 3.5 μg/ml, and as for the Poncirus trifoliata seed extract, the 50% antiviral effect (EC.sub.50) was showed at 0.2 μg/ml or more. In addition, as a result of observing the cells by a microscope, as for Tamiflu, many cells were infected and dead, while even the treatment with 0.07 μg/ml Poncirus trifoliata seed extract showed an anti-viral effect (FIG. 2a). In the results with respect to the H5N1 infected strain, as for Tamiflu, the EC.sub.50 value was observed at 1.5 μg/ml, and as for the Poncirus trifoliata seed extract, the 50% antiviral effect (EC.sub.50) was showed at 0.5 μg/ml (FIG. 2b). In the results with respect to the H3N2 infected strain, as for Tamiflu, the EC.sub.50 value was observed at 3.5 μg/ml, and as for the Poncirus trifoliata seed extract, the 50% antiviral effect (EC.sub.50) was showed at 1.5 μg/ml (FIG. 2c). In the results with respect to the influenza B virus infected strain, as for both Tamiflu and the Poncirus trifoliata seed extract, the 50% antiviral effect was showed at 13.7 μg/ml (FIG. 2d).

[0073] Overall, the above results show that the Poncirus trifoliata seed extract have an antiviral activity, which is as high as that of Tamiflu, on influenza A viruses H1N1, H3N2, and H5N1, and influenza B virus. These results suggest the possibility that the Poncirus trifoliata seed extract has a relatively high treatment effect on influenza viruses.

[0074] Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.