Composition having inhibitory effect on virus and bacteria

Abstract

The present invention relates to an antimicrobial composition comprising a grape extract, a lemon extract, and a lavender extract. Not only providing excellent antiviral, antibacterial, and antifungal effects but also being derived from natural products, the composition of the present invention can be used safely without toxicity and side effects and can find various applications in medicinal products, foods, cosmetic products, quasi-drugs, etc.

Claims

1. A method for treatment or prevention of infection with Enterovirus 71 or diseases caused by the infection with Enterovirus 71, comprising: administering an antimicrobial composition comprising a grape extract, a lemon extract and a lavender extract as active ingredients to a subject in need thereof, wherein the antimicrobial composition comprises the grape extract, the lemon extract and the lavender extract in a weight ratio of 1-2:1-2:1-2.

2. A method for inhibiting activity or infection of Enterovirus 71, comprising: applying an antimicrobial composition comprising a grape extract, a lemon extract and a lavender extract as active ingredients to an area in need thereof, wherein the antimicrobial composition comprises the grape extract, the lemon extract and the lavender extract in a weight ratio of 1-2:1-2:1-2.

3. The method of claim 1, wherein the grape extract is obtained from grape skin.

4. The method of claim 1, wherein the lavender extract is lavender oil.

5. The method of claim 2, wherein the grape extract is obtained from grape skin.

6. The method of claim 2, wherein the lavender extract is lavender oil.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1 is a graph indicating the inhibitory effect on enterovirus 71 for each active ingredient and mixture containing the active ingredients in various ratios.

(2) FIG. 2 is a Western blot result showing the levels of VP proteins of enterovirus 71 in the case of treatment with the compositions of the present invention.

EXAMPLES

(3) In the following, exemplary embodiments of the inventive concept will be explained in further detail with reference to examples. However, the following examples are meant to exemplify the present invention, and the scope of the invention is not restricted by these examples.

Example 1. Preparation of Grape Extract

(4) 30 g of dried grape skin was immersed in 570 g of water, and then subjected to hydrothermal treatment (under reflux) in an extraction apparatus having a cooling condenser under the conditions of 60° C. for 1 hour. After completion of hydrothermal treatment (under reflex), the treated grape skin was centrifuged using a centrifuge (Supra 22K, Hanil, Korea) under the conditions of 8,000 rpm and 15° C. for 20 minutes to separate an extract. The separated extract was filtered with 0.8 μm and 0.45 μm filters to obtain a filtrate. The filtrate was concentrated using a concentration apparatus (Rotary Evaporator NE-series, Eyela, Japan) under the conditions of 60° C. and 30 hPa for 2 hours. The concentrate was prelyophilized under the conditions of −21° C., and then lyophilized using a lyophilizer (Bondiro, Ilshin, Korea) at −80° C. for 48 hours to obtain an extract. The resultant grape extract was referred to as “Sample 1” and used in the following test examples.

Example 2. Preparation of Lemon Extract

(5) 30 g of dried lemon was immersed in 570 g of water, and then the same method as described in Example 1 above was used to obtain a lemon extract. The resultant lemon extract was referred to as “Sample 2” and used in the following test examples.

Example 3. Preparation of Lavender Extract

(6) 30 g of dried lavender was put in a steam distillation extraction set containing 570 g of water, and then subjected to steam distillation extraction in an essential oil extraction apparatus having a cooling condenser under the conditions of 100° C. for 1 hour. The steam distillation extract obtained by cooling was cooled at 5° C. and left to stand for 24 hours to obtain separated oil from the upper layer. The resultant lavender oil was referred to as “Sample 3” and used in the following test examples.

Example 4. Preparation of Mixture Compositions of Grape Extract, Lemon Extract and Lavender Extract

(7) The grape extract, lemon extract and lavender extract obtained in Examples 1, 2 and 3 above, respectively, were mixed in weight ratios of 1:1:1, 2:1:1, 1:2:1 and 1:1:2 using a homogenizer (IKA T25, IKA, JPN) to obtain four mixture compositions. The resultant mixture compositions were referred to as “Sample 4,” “Sample 5,” “Sample 6,” and “Sample 7” in the order of the weight ratios of 1:1:1, 2:1:1, 1:2:1 and 1:1:2, respectively, and used in the following test examples.

Test Example 1. Inhibitory Effect on Enterovirus 71 Infection

(8) 1-1. Measurement of Antiviral Activity Using SRB Assay

(9) In order to confirm the inhibitory effect of the composition according to the present invention on enterovirus 71 infection, the test was conducted by applying sulforhodamine B (SRB) assay (Choi et al, Antiviral activity of raoulic acid from Raoulia australis against Picornaviruses, Phytomedicine, 2009).

(10) Vero cells were seeded into well-plates at a concentration of 3×10.sup.4 cell/well and cultured for 24 hours using DMEM media containing 10% FBS. After removing the media and washing with PBS, enterovirus 71 suspension and FBS media were added to wells containing the samples prepared in Examples above at a concentration of 100 μg/mL, and then the cells were cultured in a CO.sub.2 incubator (Sanyo, JPN) in 5% CO.sub.2 condition for two days. Thereafter, they were washed with PBS, and 100 μl of 70% acetone was added to each well, left at room temperature for 30 minutes, and then removed. After drying the well using a dry oven (Hanbaek, KOR) for 10 minutes, 100 μl of 0.4% SRB dye containing 1% acetic acid was added thereto and left at room temperature for 30 minutes. The SRB was removed, and the plates were washed several times with cosolvents containing 1% acetic acid, followed by drying again using a dry oven. A phase-contrast microscope (Nikkon, JPN) was used for observation. Each dyed well was dissolved with Tris-base solution and left. Then, optical density (OD) values were measured at 560 nm using an ELISA reader (EPOCH2, Biotek, USA).

(11) The optical density (OD) values as measured were included in the following Equation 1 to calculate the antiviral activity rates of Samples 1 to 7 in percentage. Here, the antiviral activity rate means the activity that the samples inhibit the growth and proliferation of enterovirus 71, which may be also construed that the activity rate means the rate of cell protection against enterovirus 71 infection.

(12) $\begin{array}{cc}\frac{\begin{array}{c}\mathrm{OD}(\mathrm{Experimental}\mathrm{group}\mathrm{treated}\mathrm{wtih}\mathrm{virus}+\\ \mathrm{sample})-\mathrm{OD}(\mathrm{Control}\mathrm{group}\\ \mathrm{treated}\mathrm{wtih}\mathrm{virus})\end{array}}{\begin{array}{c}\mathrm{OD}\left(\mathrm{Control}\mathrm{group}\mathrm{untreated}\mathrm{with}\mathrm{virus}\right)-\\ \mathrm{OD}\left(\mathrm{Control}\mathrm{group}\mathrm{treated}\mathrm{wtih}\mathrm{virus}\right)\end{array}}\times 100=\mathrm{antiviral}\mathrm{activity}\mathrm{rate}\left(\%\right)& \left[\mathrm{Equation}1\right]\end{array}$

(13) The antiviral activity rate (%) of each composition, calculated according to Equation 1 above, is shown in graph of FIG. 1. As can be seen from FIG. 1, it was confirmed that the compositions comprising the respective single extracts alone (Samples 1, 2 and 3) have little cell protective effect against enterovirus 71 infection. In contrast, it could be confirmed that all of the mixture compositions (Samples 4, 5, 6 and 7) show significant cell protective effects against enterovirus 71 infection, irrespective of the mixing ratios. Particularly, it was observed that the composition (Sample 4) in which the grape extract, lemon extract and lavender extract are mixed in a ratio of 1:1:1 exhibits the most excellent antiviral activity.

(14) 1-2. Confirmation of Antiviral Activity Through Western Blot Analysis of VP Proteins

(15) In order to re-verify the antiviral efficacy of the compositions according to the present invention, Western blotting was performed for VP0 capsid protein of enterovirus 71.

(16) The supernatant containing VP0 capsid protein of enterovirus 71 was subjected to electrophoresis on acrylamide gels, and then moved to iBlot Transfer Stack, PVDF, regular size (IB401001, invitrogen). The membranes were reacted with 5% skim milk (232100, Difco) at room temperature for 1 hour, and then washed 3 times with phosphate buffered saline (PBS) Tween-20.

(17) Thereafter, a mouse anti-enterovirus 71 monoclonal antibody (MAB 979, Millipore) as a primary antibody was reacted with the membranes at room temperature for 2 hours, and then washed several times with PBS Tween-20. For α-tubulin, a mouse IgG1 monoclonal antibody (SC-32293, Santa Cruz) was used as a primary antibody.

(18) As a secondary antibody for detecting VP proteins of enterovirus 71 and α-tubulin, polyclonal goat anti-mouse IgG(H+L) HRP (SA001-500, GenDEPOT) was used. The polyclonal goat anti-mouse IgG(H+L) HRP was diluted 5,000 times in 5% skim milk, reacted with the membranes at room temperature for 1 hour, and washed with PBS Tween-20, to perform the Western blot.

(19) As can be seen from FIG. 2, α-tubulin was detected in all the control group untreated with virus, control group treated with virus and experimental groups treated with virus+sample. VP0 capsid protein of enterovirus 71 was not shown in the control group untreated with virus, whereas the proteins were very distinctly shown in the control group treated with virus. VP0 capsid protein of enterovirus 71 was hardly observed in the experimental group treated with Sample 4 falling under the present invention. In addition, VP0 capsid protein of enterovirus 71 was partially detected in the experimental groups treated with Sample 5, 6 or 7 falling under the present invention, but observed at very low detection levels as compared with the control group treated with virus.

(20) Accordingly, it was demonstrated that when the compositions according to the present invention are treated against viruses, significant levels of antiviral activity are exhibited. Particularly, the composition in which the grape extract, lemon extract and lavender extract are mixed in a ratio of 1:1:1 exhibits the most excellent antiviral activity.

Test Example 2. Antiviral Effects Against Influenza a Virus

(21) In order to test the antiviral effects of the compositions according to the present invention, the experiments on Influenza A virus were conducted by the Virus Research and Testing Center of Korea Research Institute of Chemical Technology, which is a Korean government-funded research institute.

(22) As for the control group in this experiment, Influenza A virus (H3N2) and water were mixed in a volume ratio of 1:9 and reacted at room temperature (approximately 23° C.). Each virus titer was measured after 5 minutes and 60 minutes to determine the Virus reduction.

(23) As for the experimental group, Influenza A virus (H3N2) and the composition of the present invention in the form of liquid (Sample 4) were mixed in a volume ratio of 1:9 and reacted at room temperature (approximately 23° C.). Each virus titer was measured after 5 minutes and 60 minutes to determine the Virus reduction.

(24) TABLE-US-00001 TABLE 1 Virus titer (CCID.sub.50/well) Virus reduction (%) After After After After 5 min 60 min 5 min 60 min reaction reaction reaction reaction Control Group 2,658,000 2,658,000 — — Experimental <316 <316 >99.988% >99.988% Group

(25) As can be confirmed from Table 1, the composition of the present invention made more than 99.9% of Influenza A virus to be inactive even within a very short period of time such as five minutes, thereby proving its strong antiviral effects.

Test Example 3. Antibacterial and Antifungal Effects

(26) In order to test an antibacterial activity and an antifungal activity of the samples obtained in Examples above, experiments according to agar serial dilution method and paper disc method were conducted.

(27) A total of 5 types of strain, Staphylococcus aureus (KCTC 6910) as Gram-positive bacteria, Pseudomonas aeruginosa (KCTC 1637) and E. Coli (KCTC 1039) as Gram-negative bacteria, Candida albicans (KCTC 7965) as yeasts and Aspergillus niger (KCTC 6910) as filamentous fungi, were used, and they were all sold from the Korea Research Institute of Bioscience and Biotechnology. Specific experimental methods and results are as follows.

(28) 3-1. Experiment According to Agar Serial Dilution Method

(29) The antimicrobial activity of the compositions was confirmed by measuring the minimum inhibitory concentration according to agar serial dilution method.

(30) In order to conduct antimicrobial tests, the bacteria was inoculated into a tryptic soy agar medium to preincubate at 37° C. for 24 hours; the yeasts was inoculated into a potato dextrose agar medium to preincubate at 25° C. for 2 days; and the filamentous fungi was inoculated into a potato dextrose agar medium to preincubate at 25° C. for 7 days, and then the spores of filamentous fungi formed on the surface of the medium were collected using a spreader and diluted in sterile saline solution for use.

(31) 2 mL of the composition diluted with 5% dimethyl sulfoxide (DMSO) physiological saline solution was added to each sterilized Petri dish. 5% DMSO physiological saline solution was used as a control group. 18 mL of each tryptic soy agar medium and potato dextrose agar medium sterilized was added to each Petri dish, followed by stirring, and then left to stand to be coagulated.

(32) Thereafter, each strain preincubated was spread on Petri dish (spread cell concentration: about 1×10.sup.6 CFU/mL of bacteria; about 1×10.sup.5 CFU/mL of yeasts; and about 1×10.sup.4 CFU/mL of filamentous fungi). The bacteria were incubated at 37° C. for 24 hours, the yeasts were incubated at 25° C. for 3 days, and the filamentous fungi were incubated in a 25° C. incubator for 7 days. Thereafter, the formation of colonies in each compartment was observed.

(33) The minimum sample concentration (i.e., minimum inhibitory concentration (MIC)) of the not-grown plates was determined, and the results are shown in Table 2 below. Here, the smaller MIC value indicates the higher antimicrobial effect.

(34) TABLE-US-00002 TABLE 2 MIC (μg/mL) Sample 4 Sample 5 Sample 6 Sample 7 Strain Sample 1 Sample 2 Sample 3 (mixing ratio 1:1:1) (mixing ratio 2:1:1) (mixing ratio 1:2:1) (mixing ratio 1:1:2) S. aureus >5,000 5,000 500 100 150 100 200 P. aeruginosa >5,000 >5,000 1,000 100 200 150 150 E. Coli >5,000 >5,000 750 75 200 100 100 C. albicans >5,000 >5,000 1,000 50 100 150 100 A. niger >5,000 >5,000 2,000 200 250 300 200

(35) As can be confirmed from Table 2, all the mixture compositions of the present invention exhibited strong antimicrobial effects on various kinds of bacteria, yeasts and filamentous fungi as compared with the single extracts.

(36) 3-2. Experiment According to Paper Disc Method

(37) In order to test the antimicrobial effect of the compositions, experiments according to paper disc method were conducted.

(38) The strain incubated in each plate medium was taken in an amount of one platinum loop and incubated in 10 mL liquid medium for 24 hours for activation. 0.1 mL of the microbial solution incubated in the 10 mL liquid medium was further incubated for 6 hours, and then the microbial solution was inoculated into each plate medium so as to be about 10.sup.7 CFU/mL, and uniformly spread. Thereafter, sterile paper discs (6 mm, Satorius, Germany) were placed on solid plate media, and then the samples dissolved in a solvent were absorbed to be 0.05 ml/disk and incubated. Staphylococcus aureus, Pseudomonas aeruginosa and E. Coli were incubated at 37° C.; and Candida albicans and Aspergillus niger, which are types of fungus, were incubated at 27° C. for 24 hours and 120 hours, respectively. And then, the diameters of the transparent zones around the paper disks were measured.

(39) The results of measurement are shown in Table 3 below. Here, the transparent zone around the paper disk is an index indicating the inhibition degree of proliferation of the corresponding strain. Accordingly, the greater diameter (mm) of the transparent zone indicates the higher antimicrobial effect on the strain.

(40) TABLE-US-00003 TABLE 3 Transparent zone diameter (mm) Sample 4 Sample 5 Sample 6 Sample 7 Strain Sample 1 Sample 2 Sample 3 (mixing ratio 1:1:1) (mixing ratio 2:1:1) (mixing ratio 1:2:1) (mixing ratio 1:1:2) S. aureus — 8 15 24 22 23 18 P. aeruginosa — — 12 24 21 22 20 E. Coli — — 13 30 20 25 20 C. albicans — — 11 35 22 20 20 A. niger — — 8 22 18 16 18

(41) As shown in Table 3, Sample 1 and Sample 2, which are single extracts, have little antimicrobial effect, and Sample 3 partially exhibits antimicrobial effect. On the other hand, all the mixture compositions show significantly excellent antimicrobial effects on various kinds of bacteria, yeasts and filamentous fungi as compared with the single extracts. Particularly, it was confirmed that Sample 4 at a mixture ratio of 1:1:1 exhibits the most excellent antibacterial and antifungal effects.

(42) Hereinafter, toner and lotion are provided as formulation examples of the present invention. However, these should not be construed as limiting the formulation of the composition according to the present invention. Additionally, a person having ordinary skill in the art may make any modifications within the scope of the present invention.

(43) TABLE-US-00004 TABLE 4 Formulation example 1. Toner Ingredient Content (%) 1 Purified water residual quantity 2 Butylene glycol 5.00 3 Composition of the present invention 2.00 4 Glycerine 4.00 5 Disodium EDTA 0.02 6 Carbomer 0.10 7 Betaine 0.50 8 Hyaluronic acid 0.01 9 Ethanol 4.00 10 PEG-60 hydrogenated castor oil 0.30 11 Methylparabene 0.10 12 Phenyl trimethicone 0.05 13 Fragrance ingredient trace 14 Coloring agent trace

(44) TABLE-US-00005 TABLE 5 Formulation example 2. Lotion Ingredient Content (%) 1 Purified water residual quantity 2 Methylparabene 0.20 3 Composition of the present invention 5.00 4 Glycerine 5.00 5 Butylene glycol 7.00 6 Carbomer 0.10 7 Xantan gum 0.05 8 Cetearyl alcohol 3.00 9 Glyceryl stearate 0.50 10 Propylparaben 0.10 11 Mineral oil 3.00 12 Plant squalene 3.00 13 Cetyl ethylhexanoate 3.00 14 Arginine 0.10 15 Fragrance trace

(45) TABLE-US-00006 TABLE 6 Formulation example 3. Cream Ingredient Content (%) 1 Purified water residual quantity 2 Methylparabene 0.20 3 Composition of the present invention 5.00 4 Glycerine 5.00 5 Butylene glycol 7.00 6 Carbomer 0.20 7 Xantan gum 0.08 8 Cetearyl alcohol 3.00 9 Glyceryl stearate 0.50 10 Behenyl alcohol 2.00 11 Hydrogenated lecithin 0.20 12 Propylparaben 0.10 13 Mineral oil 3.00 14 Plant squalene 3.00 15 Cetyl ethylhexanoate 3.00 16 Arginine 0.20 17 Fragrance trace

(46) TABLE-US-00007 TABLE 7 Formulation example 4. Essence Ingredient Content (%) 1 Purified water residual quantity 2 Methylparabene 0.20 3 Composition of the present invention 5.00 4 Hyaluronic acid 0.05 5 Butylene glycol 2.00 6 Carbomer 0.08 7 Xantan gum 0.04 8 Cetearyl alcohol 0.50 9 Glyceryl stearate 0.50 10 Propylparaben 0.10 11 Mineral oil 1.00 12 Plant squalene 2.00 13 Cetyl ethylhexanoate 1.00 14 Arginine 0.08 15 Ethanol 4.00 16 PEG-60 hydrogenated castor oil 0.30 17 Fragrance trace

(47) TABLE-US-00008 TABLE 8 Formulation example 5. Face pack Ingredient Content (%) 1 Purified water residual quantity 2 Glycerin 10.00  3 Composition of the present invention 5.00 4 Butylene glycol 5.00 5 Kaolin 4.00 6 Caprylic/capric triglyceride 4.00 7 Cyclomethicone 1.00 8 Magnesium aluminum silicate 1.00 9 PEG-100 stearate, glyceryl stearate 2.00 10 Xantan gum 0.10 11 Ethanol 3.00 12 Methylparabene 0.20 13 Chlorphenesin 0.10 14 Titanium dioxide 0.50 15 Fragrance trace