Use of Anthocyanin-Anionic Polysaccharide Complex for Preventing or Treating Influenza Virus A Infection

Abstract

The present invention relates to a use of an anthocyanin-anionic polysaccharide complex for preventing or treating influenza virus A infection and, more specifically, to a pharmaceutical composition, a food/health functional food/health aid food composition, and a quasi-drug composition, each comprising an anthocyanin-anionic polysaccharide complex as an active ingredient for preventing or treating influenza virus A infection diseases. A composition comprising an anthocyanin-anionic polysaccharide complex as an active ingredient provided in the present invention exhibits excellent anti-viral activity against influenza virus A and as such, can find very advantageous applications in developing an agent for prevention or treatment of diseases caused by influenza virus A infection.

Claims

1. A pharmaceutical composition comprising an anthocyanin-anionic polysaccharide complex as an active ingredient for the prevention or treatment of influenza virus A infection disease.

2. The pharmaceutical composition according to claim 1, wherein the anionic polysaccharide contains a sulfate group or a carboxyl group.

3. The pharmaceutical composition according to claim 1, wherein the anionic polysaccharide is at least one selected from the group consisting of alginic acid, hyaluronic acid, o-sulfated hyaluronic acid (o-sulfated HA), dextran sulfate, chondroitin sulfate, dermatan sulfate, keratin sulfate, heparin, heparin sulfate, fucoidan, carrageenan, mixtures thereof, and complexes thereof.

4. The pharmaceutical composition according to claim 1, wherein the anthocyanin is at least one selected from the group consisting of peonidin, cyanidin 3-arabinoside, cyanidin-3-(xylosylglucose)-5-galactose, cyanidien-3-xyloside, cyanidin3-glucoside, cyanidin 3-galactoside, cyanidin-3-(coumaroyl-xylosylglucose)-5-galactose, delphinidin 3-glucoside, delphinidin 3-rutinoside, peonidin 3-arabinoside, peonidin 3-galactoside, petunidin 3-glucoside, cyanidin, delphinidin, malvidin, pelargonidin, peonidin, cyanidin 3,5-diglucoside, cyanidin 3-rutinoside, pelargonidin 3-glucoside, peonidin 3-glucoside, malvidin 3-glucoside, and malvidin 3,5-diglucoside.

5. The pharmaceutical composition according to claim 1, wherein the anthocyanin-anionic polysaccharide complex is prepared by a method comprising the following steps: (a) forming an anthocyanin-anionic polysaccharide complex in acidic environment; and (b) recovering the complex formed above.

6. The pharmaceutical composition according to claim 1, wherein the influenza virus A is H1N1 subtype.

7. The pharmaceutical composition according to claim 1, wherein the influenza virus A infection disease is flu, cold, laryngopharyngitis, bronchitis, pneumonia, bird flu, swine flu or goat flu.

8. The pharmaceutical composition according to claim 1, wherein the weight ratio of anthocyanin and anionic polysaccharide in the complex is 1:1 to 100.

9. A food composition, comprising the pharmaceutical composition according to claim 1 incorporated into an edible food product.

10. A drug delivery system, the pharmaceutical composition according to claim 1 disposed in a quasi-drug composition.

11. The drug delivery system according to claim 10, wherein the quasi-drug is a gas phase filter, filter coating agent, hand wash, disinfectant cleaner, shower foam, wet tissue, detergent soap, humidifier filler, mask or air freshener.

12. A method for preventing or treating disease caused by influenza virus A infection, the method comprising administering to a mammal infected with influenza virus A a therapeutically effective amount of the pharmaceutical composition according to claim 1.

13.-14. (canceled)

15. The method according to claim 12, wherein the disease is selected from the group consisting of flu, cold, laryngopharyngitis, bronchitis, pneumonia, bird flu, swine flu, and goat flu.

16. A method of treating an influenza virus A infection, the method comprising administering a therapeutically effective amount of the pharmaceutical composition according to claim 1 to a mammal infected with influenza virus A.

17. The method according to claim 16, wherein the anionic polysaccharide: (i) contains a sulfate group or a carboxyl group; and/or (ii) is at least one selected from the group consisting of alginic acid, hyaluronic acid, o-sulfated hyaluronic acid (o-sulfated HA), dextran sulfate, chondroitin sulfate, dermatan sulfate, keratin sulfate, heparin, heparin sulfate, fucoidan, carrageenan, mixtures thereof, and complexes thereof.

18. The method according to claim 16, wherein the anthocyanin is at least one selected from the group consisting of peonidin, cyanidin 3-arabinoside, cyanidin-3-(xylosylglucose)-5-galactose, cyanidien-3-xyloside, cyanidin3-glucoside, cyanidin 3-galactoside, cyanidin-3-(coumaroyl-xylosylglucose)-5-galactose, delphinidin 3-glucoside, delphinidin 3-rutinoside, peonidin 3-arabinoside, peonidin 3-galactoside, petunidin 3-glucoside, cyanidin, delphinidin, malvidin, pelargonidin, peonidin, cyanidin 3,5-diglucoside, cyanidin 3-rutinoside, pelargonidin 3-glucoside, peonidin 3-glucoside, malvidin 3-glucoside, and malvidin 3,5-diglucoside.

19. The method according to claim 16, wherein the influenza virus A is H1N1 subtype.

20. The method according to claim 16, wherein the weight ratio of anthocyanin to anionic polysaccharide in the complex is 1:1 to 100.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0099] FIG. 1 is a set of graphs showing the results of measuring the size of anthocyanin-alginic acid complex particles using a DLS device (ATC: anthocyanin, ATC/Alg complex: anthocyanin-alginic acid complex).

[0100] FIG. 2 is a set of graphs showing the results of confirming the formation of anthocyanin-alginic acid complex particles using a spectrophotometer (ATC: anthocyanin, ATC/Alg complex: anthocyanin-alginic acid complex).

[0101] FIG. 3 is a set of graphs showing the results of confirming the stability of anthocyanin-alginic acid complex particles over time using a spectrophotometer (ATC: anthocyanin, ATC/Alg complex: anthocyanin-alginic acid complex).

[0102] FIG. 4 is a graph showing the results of confirming the inhibitory effect of the anthocyanin-alginic acid complex on influenza virus A (H1N1) through a plaque reduction assay.

[0103] FIG. 5 is a graph showing the results of confirming the inhibitory effect of the anthocyanin-alginic acid complex on influenza virus B through a plaque reduction assay.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0104] Hereinafter, the present invention will be described in detail by the following examples and experimental examples.

[0105] However, the following examples and experimental examples are only for illustrating the present invention, and the contents of the present invention are not limited thereto.

Example 1: Preparation of Anthocyanin-Anionic Polysaccharide Nanocomplex

[0106] Anthocyanin was obtained by crushing the raw fruit of aronia, adsorbing the juice to a polyphenol adsorption resin, eluting thereof with an ethyl alcohol aqueous solution, and pulverizing thereof. 20 mg of the anthocyanin powder prepared above was dissolved in 5 ml of phosphate buffer (pH 3, PB 3) at 40° C., and 200 mg of alginic acid (anionic polysaccharide) was dissolved in 10 ml of deionized water (D.I water) at room temperature. An anthocyanin-anionic polysaccharide nanocomplex was prepared by adding the anthocyanin solution to the alginic acid solution (1:1, V/V), followed by stirring at room temperature for 48 hours.

Example 2: Measurement of Particle Size of Anthocyanin-Anionic Polysaccharide Nanocomplex

[0107] The particle size distribution of the anthocyanin-alginic acid nanocomplex prepared in Example 1 was measured using Zeta sizer Nano Zs (Malvern Instruments Ltd, UK), a dynamic light scattering (DLS) measuring device. As a result of measuring the anthocyanin-alginic acid nanocomplex particle size using a DLS measuring device, the complex particle size was about 360 nm. As alginic acid (−50±1.66 mV) formed a complex with anthocyanin (2.3±1.01 mV), the negative charge strength of alginic acid was decreased, and the zeta potential of the complex was −26.4±1.17 mV (FIG. 1).

Example 3: Confirmation of Anthocyanin-Anionic Polysaccharide Nanocomplex Formation

[0108] 1 ml of the complex prepared in Example 1 was put into a UV cuvette, and the maximum absorbance wavelength band of anthocyanin was confirmed through a spectrophotometer. The anthocyanin solution dissolved in PB 3/D.I water (1:1 volume) was also subjected to the same process as above, and the changes of the color of the solution and the maximum absorbance wavelength band were confirmed as the complex was formed.

[0109] As a result, it was observed that the anthocyanin-alginic acid complex was formed by the ionic bond created by the cationicity of anthocyanin and the anionicity of alginic acid, and the maximum absorbance of anthocyanin moved to a longer wavelength due to the π-π interaction between anthocyanin molecules, and thus the sample that formed the complex was slightly purple. Therefore, it was confirmed that the anthocyanin-alginic acid complex was formed (FIG. 2).

Example 4: Confirmation of Stability of Anthocyanin-Anionic Polysaccharide Nanocomplex Over Time

[0110] The amount of anthocyanin was confirmed using a spectrophotometer to confirm the stability of the complex prepared in Example 1 over time. 40 ml of the anthocyanin-alginic acid complex was centrifuged at 13000 rpm for 30 minutes, and the precipitate was dispersed in PBS (pH 3 or pH 7.4). In addition, anthocyanin was prepared by dissolving thereof in PBS (pH 3 or pH 7.4). The absorbance of the complex and anthocyanin dispersed in PBS (pH 3 or pH 7.4) was measured with a spectrophotometer at each wavelength band of 513 nm or 520 nm, respectively, and the change in the absorbance value of anthocyanin was observed.

[0111] As a result, it was confirmed that anthocyanin was stable in a low pH environment (pH 3). Specifically, after 76 hours in a low pH environment (pH 3), the complex contained 18.7% more anthocyanin and was more stable. Since decomposition occurs as the pH increases, the maximum absorbance of anthocyanin was significantly decreased within 4 hours at pH 7.4, whereas that of the complex showed a small decrease. After 4 hours, it was confirmed that anthocyanin and complex decreased at a similar rate. As a result of expressing the maximum absorbance of anthocyanin as a percentage, it was found that anthocyanin contained 42.5% less anthocyanin than the complex within 4 hours at pH 7.4. Therefore, it was confirmed that the complex has higher stability than anthocyanin (FIG. 3).

Example 5: Confirmation of Inhibitory Effect of Anthocyanin-Anionic Polysaccharide Nanocomplex on Intracellular Viral Infection

[0112] In order to confirm the antiviral effect of the anthocyanin-alginic acid complex prepared in Example 1, a plaque reduction assay for influenza virus was performed. First, a 12-well cell culture dish filled with a monolayer of MDCK cells was prepared. After mixing the prepared anthocyanin-alginic acid complex with influenza A virus (A/California/07/2009, H1N1) by concentration, 0.1 mL each was placed in a 12-well cell culture dish with MDCK cells, and the cells were infected with the virus for 1 hour and 30 minutes. As a comparative group, alginic acid and anthocyanin forming the complex were also performed in the same manner as above at the same concentration. After 1 hour and 30 minutes, 1.5 mL of 1% agarose gel containing serum-free DMEM was added to each well and hardened. After culturing the cells for 72 hours in a 37° C., 5% CO.sub.2 incubator, a cell fixative solution (methanol:acetic acid=3:1) and a crystal violet solution were mixed (1:1), and 2 mL each was put on the solid gel and stained for 24 hours. After removing and washing the gel in the well, the number of plaques, which are unstained parts, was counted. The same method was carried out with influenza B virus. As a positive control, oseltamivir was used.

[0113] A decrease in the number of plaques means a decrease in the number of viruses having infectivity.

[0114] As shown in FIG. 4, anthocyanin showed a significant antiviral effect against influenza virus A from a concentration of 1 μg/ml or higher, and alginic acid, a substance for maintaining stability of anthocyanin, did not show an antiviral effect. In the case of the anthocyanin-alginic acid complex treatment group, it was confirmed that Influenza virus A was remarkably inhibited even in the concentration combination (alginic acid 0.5 ug/ml+anthocyanin 0.1 ug/ml) that showed little effect in each single administration group. From the above results, it was confirmed that the anthocyanin-alginic acid complex exhibited a synergistic effect in the antiviral effect against influenza virus A. In particular, it was confirmed that the antiviral effect of the anthocyanin-alginic acid complex against influenza virus A was significantly superior to that of oseltamivir used as a positive control even at a lower concentration.

[0115] As shown in FIG. 5, alginic acid showed an antiviral effect at a certain concentration or higher against influenza B virus unlike influenza A virus, and anthocyanin also showed an antiviral effect of about 50% at a concentration of 1 μg/ml or higher. In the case of the anthocyanin-alginic acid complex treatment group, the synergistic effect of the combination of alginic acid and anthocyanin did not appear, and the anthocyanin-alginic acid complex treatment showed only an antiviral effect similar to that of the anthocyanin alone treatment.

INDUSTRIAL APPLICABILITY

[0116] A composition comprising an anthocyanin-anionic polysaccharide complex as an active ingredient provided in the present invention exhibits excellent anti-viral activity against influenza virus A and as such, can find very advantageous applications in developing an agent for prevention or treatment of diseases caused by influenza virus A infection.