Curcuminoid-based compound/stevioside-containing complex for the prevention and treatment of an influenza virus infection

Abstract

The present invention relates to a complex comprising a compound of formula (I), or a plant extract comprising the compound or a fraction thereof; and stevioside, or a plant extract comprising the stevioside or a fraction thereof, and relates to a pharmaceutical composition for preventing or treating an influenza virus infection comprising the complex as an active ingredient. Also, the present invention relates to a food composition for preventing or improving an influenza virus infection, a virucidal quasi-drug composition, a virucidal feed additive, and a feed, which comprises the complex as an active ingredient. According to the present invention, the complex comprising a compound of formula (I), or a plant extract comprising the compound or a fraction thereof; and stevioside, or a plant extract comprising the stevioside or a fraction thereof exhibits a virucidal effect and an effect of inhibiting cell degradation against an influenza virus as well as antiviral efficacy in a specific pathogen-free (SPF) chicken, and thus can be usefully used in the prevention and treatment of an influenza virus infection.

Claims

1. A method of treating a H9N2 influenza virus infection, comprising administering to an individual in need thereof a composition comprising therapeutically effective amounts of the compound of formula (I) and stevioside: ##STR00008## wherein, R.sup.1 and R.sup.2 are each independently hydrogen, hydroxy, or C.sub.1-C.sub.10 alkoxy; n is an integer of 1 to 5; and m is an integer of 1 to 5.

2. The method of claim 1, wherein the composition is orally administered.

3. The method of claim 1, wherein the compound of formula (I) is obtained from turmeric extract, and the stevioside is obtained from Stevia rebaudiana Bertoni.

4. The method of claim 1, wherein the composition is formulated as at least one selected from a tablet, a pellet, powder, a granule, a capsule, a suspension, a liquid, an emulsion, a syrup, a sterilized aqueous solution, a non-aqueous solvent, a lyophilized preparation, and a suppository.

5. The method of claim 1, wherein the composition is a food.

Description

BEST MODE

(1) Hereinafter, the present invention will be described in detail through examples and experimental examples. However, the description proposed herein is just a preferable example for the purpose of illustrations only, not intended to limit the scope of the invention, so it should be understood that the examples are provided for a more definite explanation to an ordinary person skilled in the art.

EXAMPLE 1

Preparation of Turmeric Extract

(2) A turmeric used in this example is generally available in a medicinal herb shop or market. The steamed and dried radix of Curcuma longa Linne was purchased, and to effectively obtain an extract of the present invention, was milled in powder form. 1.6 kg of turmeric was dissolved in 7.5 l of 100% ethanol (EtOH), placed at room temperature for 5 days, and filtered through a filter paper and concentrated, to obtain 170 g of a turmeric ethanol extract.

EXAMPLE 2

Separation and Purification of Turmeric Fraction and Curcuminoid-based Compound from Turmeric Extract

(3) 170 g of the turmeric ethanol extract obtained in example 1 was suspended in 1 l of water. The suspension was put in a separatory funnel, and fractionally extracted with n-hexane and ethylacetate in sequence, to yield 23 g of an n-hexane soluble extract, 85 g of an ethylacetate soluble extract, and 34 g of a water soluble extract.

(4) 85 g of the obtained ethylacetate soluble extract was separated into 15 fractions Fr.-1 to Fr.-15 by silica gel column chromatography (500 g silica gel, mesh 70230) using solvents of chloroform, methanol, and mixture thereof (80:11:1) as mobile phases. Among them, 16 g of the sixth fraction Fr.-6 was separated into 5 fractions Fr.-6-1 to Fr.-6-5 by silica gel column chromatography (30 g, mesh 230400) using a mixed solvent of n-hexane and ethylacetate (20:11:1 (v/v)) as a mobile phase.

(5) After performing silica gel column chromatography on Fr.-6-2 and Fr.-6-3 fractions (11 g) using solvents of chloroform, methanol, and mixture thereof (80:14:1) as mobile phases, a fraction obtained was developed by preparative TLC using a mixed solvent of n-hexane and ethylacetate (4:1 (v/v)) as a mobile phase, to yield 8 g of a pure compound 1. Also, 14 g of the eighth fraction Fr.-8 was repetitively separated by silica gel column chromatography (30 g, mesh 230400) using a mixed solvent of n-hexane and ethylacetate (20:11:1 (v/v)) and a mixed solvent of chloroform and methanol (80:120:1 (v/v)) as mobile phases, to yield 0.4 g of a compound 2 and 0.2 g of a compound 3.

EXAMPLE 3

Structural Analysis of Curcuminoid-based Compound

(6) The molecular weight and molecular formula of the curcuminoid-based compounds obtained in Example 2 was confirmed using a VG high resolution GC/MS spectrometer (Election Ionization MS, Autospec-Ultima). Also, the molecular structure was confirmed through nuclear magnetic resonance analysis (Bruker AM500) using .sup.1H-NMR, .sup.13C-NMR, and 2D NMR spectroscopy materials.

(7) Based on comparing the above results with those of published papers, curcumin, demethoxycurcumin and bisdemethoxycurcumin represented by formulas (I) to (IV) were identified (Food Chem. 265-272, 2009; J. Nat. Prod. 1227-1231, 2002; J. Nat. Prod. 1531-1534, 1998; J. Agric. Food Chem. 3668-3672, 2002). The analysis results are shown below.

(8) ##STR00005##

(9) 1) Property: light orange powder (m.p. 183 C.)

(10) 2) Molecular weight: 368.3

(11) 3) Molecular formula: C.sub.21H.sub.20O.sub.6

(12) 4) .sup.1H-NMR (acetone-d.sub.6, 500 MHz) 7.62 (2H, d, J=15.80 Hz, H-4, H-4), 7.35 (2H, d, J=1.91 Hz, H-6, H-6), 6.83 (2H, H-3, H-5), 7.20 (2H, dd, J=8.3, 1.9 Hz, H-10, H-10), 6.90 (2H, d, J=8.15 Hz, H-9, H-6), 5.99 (1H, s, H-1), .sup.13C-NMR (acetone-d.sub.6, 125 MHz) 56.72, 102.01, 111.95, 116.64, 122.72, 124.25, 128.58, 141.81, 149.20, 150.44, 184.94.

(13) ##STR00006##

(14) 1) Property: orange powder (m.p. 220 C.)

(15) 2) Molecular weight: 338

(16) 3) Molecular formula: C.sub.20H.sub.18O.sub.5

(17) 4) .sup.1H-NMR (acetone-d.sub.6, 500 MHz) 7.62-7.55 (4H), 7.34 (1H), 7.18 (1H), 6.89 (3H), 6.70 (2H), 5.97 (1H), .sup.13C-NMR (acetone-d.sub.6, 125 MHz) 56.38, 101.79, 111.53, 116.30, 116.89, 122.12, 122.35, 123.98, 127.77, 128.24, 131.06, 141.13, 141.48, 148.87, 150.13, 160.64, 184, 66.

(18) ##STR00007##

(19) 1) Property: orange powder (m.p. 224 C.)

(20) 2) Molecular weight: 308

(21) 3) Molecular formula: C.sub.19H.sub.16O.sub.4

(22) 4) .sup.1H-NMR (acetone-d.sub.6, 500 MHz) 7.62-7.56 (6H), 6.91-6.87 (4H), 6.68-6.65 (2H), 5.98 (1H), .sup.13C-NMR (acetone-d.sub.6, 125 MHz) 101.82, 116.87, 122.11, 127.79, 131.06, 141.12, 160.58, 184, 62.

EXPERIMENTAL EXAMPLE 1

Determination of Neuraminidase A/Bervig_Mission/1/18 (rvH1N1) Inhibitory Activity of Turmeric Extract, Fraction, and Curcuminoid-Based Compound

(23) To determine the neuraminidase inhibitory activity of the turmeric extract, the turmeric fraction, and the curcuminoid-based compounds separated therefrom that were obtained in Examples 1 and 2 of the present invention, neuraminidase (R&D SYSTEM, 4858-NM) of a recombinant rvH1N1 influenza A virus of 1918 Spanish flu virus (A/Bervig_Mission/1/18) was used. 2-(4-Trimethylumbelliferyl)--D-N-acetyl-neuraminic acid sodium salt from Sigma was used as a substrate.

(24) The turmeric extract and its fraction of Examples 1 and 2 were dissolved in methanol. As a substrate, 2-(4-trimethylumbelliferyl)--D-N-acetyl-neuraminic acid sodium salt (final concentration 200 M) was added to 20 L of each solution. The result was mixed with 80 L of a tris buffer (pH 7.5) containing 5 mM CaCl.sub.2 and 200 mM NaCl, and reacted with 50 L of neuraminidase (final enzyme concentration 0.05 ng/L) as a zymogen at 25 C. for 10 minutes. The neuraminidase inhibitory activity was determined by measuring absorbance at 365 nm and emittance at 445 nm using a fluorescence spectroscope.

(25) The measurement results are shown in Table 1 below.

(26) TABLE-US-00001 TABLE 1 Neuraminidase inhibitory activity(IC.sub.50).sup.1) Substance A/Bervig-Mission/1/18(rvH1N1) Turmeric ethanol extract 3.1 g/mL Turmeric hexane fraction 262.0 g/mL Turmeric ethylacetate fraction 0.9 g/mL Turmeric water fraction 61.5 g/mL Curcumin 3.0 M Demethoxycurcumin 3.0 M Bisdemethoxycurcumin 6.0 M [Note] .sup.1)a result value is an average of two tests.

(27) As can be seen in Table 1 showing the determination results of neuraminidase inhibitory activity of each of the turmeric extract, fraction, and curcuminoid-based compounds according to the present invention, the turmeric ethanol extract had an IC.sub.50 value of 3.1 g/mL against influenza virus neuraminidase, the turmeric hexane fraction had an IC.sub.50 value of 262.0 g/mL, the turmeric ethylacetate fraction had an IC.sub.50 value of 0.9 g/mL, the turmeric water fraction had an IC.sub.50 value of 61.5 g/mL, curcumin and demethoxycurcumin had each an IC.sub.50 value of 3.0 M, and bisdemethoxycurcumin had an IC.sub.50 value of 6.0 M. Accordingly, it was determined through the above results that the turmeric extract, its fraction, and curcuminoid-based compounds according to the present invention had excellent neuraminidase inhibitory activity.

EXPERIMENTAL EXAMPLE 2

Determination of Influenza Virus Inhibitory Activity of Curcuminoid-based Compound Separated from Turmeric Extract, Ethylacetate Fraction, and Ethanol Extract

(28) To determine the antiviral effects of the curcuminoid-based compounds, ethylacetate fraction, and ethanol extract separated from the turmeric extract on influenza virus H1N1 (A/PR/8/34) and H9N2 (A/Chicken/Korea/MS96/96), a test below was carried out in vitro using a Madin-Darby canine kidney (MDCK, ATCC CCL-34) cell of a dog.

(29) First, MDCK cells were put in a 96-well microplate at a density of 110.sup.5/well, and incubated in a culture medium (EMEM) containing 100 units penicillin, 100 g streptomycin and 10% FBS. When the MDCK cells are grown as a monolayer, the MDCK cells were washed twice with a culture medium (EMEM) only containing antibiotic. Each of H1N1 and H9N2 strains was diluted with 100 TCID.sub.50, and put in an EP tube. The curcuminoid-based compounds, ethylacetate fraction and ethanol extract separated from the turmeric extract diluted with dimethylsulfoxide (DMSO) were put in each tube based on concentration, followed by reaction at 4 C. for 1 hour. After the lapse of 1 hour, the reacted solutions were inoculated into 3 wells of the pre-washed MDCK cells at each concentration, followed by incubation at 35 C. for 1 hour (hereinafter, referred to as a test sample). Under the same conditions, non-infected+non-administered cells (MDCK cells not infected with an H1N1 or H9N2 strain and not administered with the curcuminoid-based compound) and infected+non-administered cells (MDCK cells infected with an H1N1 or H9N2 strain and not administered with curcumin), incubated at 35 C. for 1 hour, were each set as a control and a virus control. After the lapse of 1 hour, the culture mediums of the plate were all removed, and the cells were washed once with PBS. 100 ml of a culture medium (EMEM) containing antibiotic and 10 g/mL trypsin was dispensed in each well of the cells, followed by incubation at 35 C. for 48 to 72 hours. The incubation was performed for 48 to 72 hours until the infected+non-administered cells (virus control) have cytopathic effects (CPE). The state of the cells was observed with an inverted microscope every day. After the cells were incubated for 48 to 72 hours, 10 Ml of a cell counting kit-8 (Dojin, Kumanoto, Japan, tetrazolium salt WST-8) was added to each well to determine the cell survival, followed by reaction at 35 C. for 2 hours, and the absorbance was measured at 450 nm. In this instance, the antiviral effects (Inhibition (%)) of the curcuminoid-based compounds, ethylacetate fraction, and ethanol extract separated from the turmeric extract of the present invention were calculated using the following mathematical formula (I) in comparison with the non-infected+non-administered cells (control) and the infected+non-administered cells (virus control). The results are shown in Table 2 below.

(30) $\begin{array}{cc}\mathrm{Inhibition}\left(\%\right)=\frac{\begin{array}{c}\begin{array}{c}OD\mathrm{value}\mathrm{of}\mathrm{test}\mathrm{sample}-\\ OD\mathrm{value}\mathrm{of}\mathrm{infected}+\end{array}\\ \mathrm{non}\text{-}\mathrm{administered}\mathrm{control}\end{array}}{\begin{array}{c}\begin{array}{c}OD\mathrm{value}\mathrm{of}\mathrm{non}\text{-}\mathrm{infected}+\\ \mathrm{non}\text{-}\mathrm{administered}\mathrm{control}-\end{array}\\ OD\mathrm{value}\mathrm{of}\mathrm{infected}+\\ \mathrm{administered}\mathrm{control}\end{array}}100& \mathrm{Equation}\left(I\right)\end{array}$

(31) wherein, the OD value is absorbance measured at 450 nm.

(32) Meanwhile, to determine the cell degeneration inhibitory effects of the curcuminoid-based compounds, ethylacetate fraction, and ethanol extract separated from the turmeric extract on influenza virus H1N1 (A/PR/8/34) and H9N2 (A/Chicken/Korea/MS96/96), MDCK cells were washed twice with a culture medium (EMEM) only containing antibiotic, inoculated with influenza virus (H1N1 or H9N2 strain), and incubated at 35 C. for 1 hour. After the lapse of 1 hour, the virus solution used in inoculation was completely removed, and the curcuminoid-based compounds, ethylacetate fraction, and ethanol extract separated from the turmeric extract were each inserted into the virus-inoculated MDCK cells. Subsequently, the cell degeneration inhibitory effects (Inhibition (%)) of the curcuminoid-based compounds, ethylacetate fraction, and ethanol extract separated from the turmeric extract of the present invention were determined using the above Equation (I). The results are shown in Table 2 below.

(33) TABLE-US-00002 TABLE 2 H1N1 (A/PR/8/34) H9N2 (A/Chicken/Korea/MS96/96) Substance CC.sub.50 (M).sup.a EC.sub.50 (M).sup.b SI.sup.c CC.sub.50 (M).sup.a EC.sub.50 (M).sup.b SI.sup.c Virucidal effect Tamiflu >200 18.5 >10.8 >200 <1 >200 Curcumin 94.1 7.1 13.3 94.1 18.6 5.1 Demethoxycurcumin 97.0 8.0 23.1 97.0 18.2 5.3 Bisdemethoxycurcumin >200 28.1 >7.1 >200 >200 <1 Ethylacetate fraction 82.1 g/mL 9.0 g/mL 9.1 82.1 g/mL 20.0 g/mL 4.1 Ethanol extract 55.7 g/mL 8.7 g/mL 6.4 55.7 g/mL 30.4 g/mL 1.8 Cell degeneration inhibitory effect Tamiflu >200 3.0 >66.7 >200 <1 >200 Curcumin 94.1 40.7 2.3 94.1 10.9 8.6 Demethoxycurcumin 97.0 60.8 1.6 97.0 24.9 3.9 Bisdemethoxycurcumin >200 141.5 >1.4 >200 181.6 >1.1 Ethylacetate fraction 82.1 g/mL 23.5 g/mL 3.5 82.1 g/mL 11.6 g/mL 7.1 Ethanol extract 55.7 g/mL 27.8 g/mL 2.0 55.7 g/mL 31.5 g/mL 1.8 [Note] .sup.aCC.sub.50, 50% cytotoxic concentration .sup.bEC.sub.50, 50% antiviral concentration .sup.cSI, selective index, CC.sub.50/EC.sub.50

(34) a. The curcuminoid-based compounds, ethylacetate fraction and ethanol extract were each mixed with virus, followed by reaction at 4 C. for 1 hour, and MDCK cells were infected with the virus. After the lapse of 1 hour, the cells were washed once with PBS and a culture medium (EMEM) containing 10 mg/mL trypsin was dispensed. The cells were incubated at 35 C. for 48 to 72 hours.

(35) b. After 1 hour of the virus infection, the medium including the virus was removed and replaced with a fresh medium containing each of the curcuminoid-based compounds, ethylacetate fraction, and ethanol extract, and then the cells were incubated for 48 to 72 hours.

(36) c. A value of CC.sub.50/EC.sub.50 as selective index (SI)

(37) As shown in Table 2, the curcuminoid-based compounds had excellent virucidal effects on an H1N1 strain exhibiting a selective index (SI) of 13.3, 23.1, and greater than 7.1. Also, the curcuminoid-based compounds exhibited a selective index (SI) of 5.1, 5.3, and less than 1.0 against an H9N2 strain. Accordingly, it was determined that the curcuminoid-based compounds had virucidal effects on a variety of virus strains. The turmeric ethylacetate fraction and turmeric ethanol extract had excellent virucidal effects on an H1N1 strain, exhibiting a selective index (SI) of 9.1 and 6.4, respectively. According to the observation results of the cell morphology with an inverted microscope, it was found that MDCK cells inoculated with virus (H1N1 or H9N2) were almost degenerated, leading to 90 to 100% of cytopathic effects, while virus-infected MDCK cells treated with the curcuminoid-based compounds, ethylacetate fraction, and ethanol extract exhibited a similar aspect to a non-infected+non-administered control.

(38) Furthermore, the curcuminoid-based compounds had cell degeneration inhibitory effects on H1N1 and H9N2. In particular, the curcuminoid-based compounds had excellent cell degeneration inhibitory effects on an H9N2 strain exhibiting a selective index (SI) of 8.6, 3.9, and greater than 1.1, and had cell degeneration inhibitory effects on an H1N1 strain exhibiting a selective index (SI) of 2.3, 1.6, and greater than 1.4. According to the observation results of the cell morphology with an inverted microscope, it was found that MDCK cells inoculated with virus (H1N1 or H9N2) were almost degenerated, leading to 90 to 100% of cytopathic effects, while virus-infected MDCK cells treated with the curcuminoid-based compounds, ethylacetate fraction, and ethanol extract exhibited a similar aspect to a non-infected+non-administered control.

(39) Accordingly, the composition of the present invention has virucidal effects by directly working on the virus before the virus infects cells, and has excellent cell degeneration inhibitory effects by preventing the virus from releasing from cells through replication after the cells are infected with the virus, and thus can be useful in preventing and treating an influenza virus infection.

EXPERIMENTAL EXAMPLE 3

Determination of Antiviral Activity of the Complex According to the Present Invention Using SPF Chicken as an Animal Model

(40) To determine the antiviral activity of the complex of the present invention which comprises a compound of formula (I) having an antiviral activity as confirmed in the above Experimental Example, a turmeric extract comprising the compound, a fraction of the extract, and stevioside, the following test was performed.

(41) First, curcumin was mixed with stevioside to obtain a complex. Specifically, 10 g/L of curcumin and 1 to 100 g/L of stevioside were well mixed and subject to ultrasonication at 70 C. for 30 minutes, and the sonicated mixture was brought into reaction in a 700W oven for 15 minutes, and cooled. The reaction for 15 minutes in the oven was further repeated 3 times.

(42) As a result, the resulting solution had the maximum 3.5 g/L of curcumin in 100 g/L of the stevioside solution, and the resulting solution was used directly or after diluting depending on its uses. The complex prepared above was confirmed to provide curcumin in an amount of 0.3 mg/kg/day.

(43) In order to confirm the antiviral and virucidal activity of the complex, 60 chickens of being specific pathogens free (SPF) and aged 3 weeks were grouped into the total six groups (10 chickens per a group), i.e., 3 test groups orally administered with curcumin, a turmeric extract, and a composition comprising a mixture of curcumin and stevioside, respectively; a test group orally administered with 10% stevioside; a challenge positive control group inoculated; and a negative control group (Table 3).

(44) TABLE-US-00003 TABLE 3 Group Samples 1 Curcumin (1 mg/kg/day) 2 Turmeric ethanol extract (comprising 10% curcumin) 3 Composition of curcumin (1%) + stevioside (10%) (300 L/dose) 4 Stevioside (10%) (300 L/dose) 5 Challenge positive control group 6 Negative control group

(45) As a test virus, influenza virus type A (H9N2) was used and the listed samples were orally administered in an amount of 10.sup.6.0/100 L/dose in each group e4 hours before the challenge inoculation of the virus to 5 days after the inoculation. After 5 days from the inoculation, a degree of inhibiting the inoculated virus proliferation was confirmed in the trachea and cecal tonsil of each group, and the results thereof are shown in Table 4.

(46) TABLE-US-00004 TABLE 4 Number Virus Mean virus of re-isolation.sup.A titer(log.sub.10EID.sub.50/g) inoculated Cecal Cecal Group chickens Trachea Tonsil Trachea Tosnil G1 10 4/10 6/10 1.7 2.5 4.2 3.6 G2 10 9/10 6/10 4.8 1.56 4.20 3.61 G3 10 8/10 2/10 4.0 2.17 1.40 2.95* G4 10 10/10 8/10 5.80 1.3 5.30 3.5 G5 10 10/10 8/10 5.70 1.9 5.40 2.88 G6 10 0/10 0/10 0.00 0.0 0.00 0.0 .sup.ANumber of virus-positive chicken/Number of inoculated chickens, *P < 0.05 by one-tailed t-test

(47) As can be seen in Table 4, when the samples of the present invention were administered 4 hours before the challenge inoculation of the virus to 5 days after the inoculation to confirm their effect on the inhibition of an influenza virus proliferation, in the trachea and cecal tonsil of the groups orally administered with curcumin, a turmeric extract, and a composition comprising a mixture of curcumin and stevioside, respectively, a degree of virus re-isolation and a mean virus titer were substantially reduced as compared with the challenge positive control group and the negative control group.

(48) Specifically, in the case of the group administered with curcumin, a degree of virus re-isolation and a mean virus titer were substantially reduced in the trachea, but were not surprisingly reduced in the cecal tonsil, as compared with the challenge positive control group. Also, in the case of the group administered with a turmeric ethanol extract comprising 10% curcumin, a degree of virus re-isolation and a mean virus titer were not surprisingly reduced in both trachea and cecal tonsil, as compared with the challenge positive control group. In contrast, in the case of the group administered with a composition comprising a mixture of curcumin and stevioside, a degree of virus re-isolation and a mean virus titer were surprisingly reduced in both trachea and cecal tonsil, as compared with the challenge positive control group, the group administered with curcumin, and the group administered with the turmeric extract.

(49) From these results, it was confirmed that the composition comprising a mixture of curcumin and stevioside can exhibit antiviral effects against viruses present in trachea, as well as cecal tonsil which is the end of internal organs when it is administered in an animal model, and therefore, it can maintain an antiviral activity in the whole body of an individual infected with a virus.

EXPERIMENTAL EXAMPLE 4

Acute Toxicity Test of Compound of the Present Invention

(50) To determine the acute toxicity of the compounds of the present invention, the following test was performed.

(51) Specific pathogens free (SPF) C57BL/6J mice including 12 female and 12 male, aged 6 weeks (sample), were grouped into four groups (3 female mice and 3 male mice as a test group), and bred in an animal room under the conditions of a temperature of 223 C., a humidity of 5510%, and an illumination strength of 12L/12D. Before the test, the mice were gone through domestication for 1 week. The mice were freely fed with laboratory animal feeds (feed for mice and rats, CJ Corporation in Seoul, Republic of Korea) and sterile water.

(52) A sample was prepared by adding 0.5% of TWEEN80 to each of the compounds of formulas (II) to (IV) obtained in Example 2 at a concentration of 50 mg/mL, and administered in a dosage of 0.04 mL (100 mg/kg), 0.2 mL (500 mg/kg), and 0.4 mL (1,000 mg/kg) per 20 g of the mouse weight. The sample was orally administered in a single dose, and after administration, side effect or survival was observed for 7 days. That is, on the day of administration, the general symptomatic change and survival were observed after 1 hour, 4 hours, 8 hours, and 12 hours of administration, and from the next day of administration to the seventh day, were observed once or more each in the forenoon and afternoon every day.

(53) The acute toxicity test results showed that all the mice administered with the sample did not have a noteworthy clinical sign and there was no dead mouse. Also, the toxicity change was not observed in the weight change, blood test, biochemical examination of blood, and autopsy findings.

(54) The curcuminoid-based compound used in the present invention is not subject to a toxicity change in all the mice until a dosage is 1,000 mg/kg, and it was confirmed that the compounds of the present invention are a stable substance having 1,000 mg/kg or more of an oral LD.sub.50, that is, a minimum lethal dose. Also, the stevioside used in the present invention is a safe substance which has been conventionally used as a sweetener. Therefore, the composition of the present invention, which comprises the curcuminoid-based compound, or a plant extract comprising the compound or a fraction thereof; and stevioside, or a plant extract comprising the stevioside or a fraction thereof, can be safely and effectively used in the treatment of an influenza virus infection.