WO2015178700A1 - Anti-viral composition comprising fermentation product of fruit of genus citrus as active ingredient - Google Patents

Abstract

The present invention relates to an anti-virus composition comprising a fermentation product of fruits of the genus Citrus as an active ingredient. Specifically, the fermentation product of fruits of the genus Citrus according to the present invention is not toxic to the human body and has an excellent inhibitory effect on proliferation of various types of influenza viruses, and thus can be used as a pharmaceutical composition having an anti-viral activity, or as a health food and a feed additive for the above purpose.

Description

Antiviral composition comprising bilberry fruit fermentation as an active ingredient

The present invention relates to a composition for the prevention and treatment of viral diseases comprising bilberry fruit fermentation as an active ingredient.

More specifically, the present invention relates to a composition for the prevention and treatment of a disease infected by one or more viruses selected from the group consisting of influenza viruses, a disease infected by a rotavirus, or a disease infected by a corona virus.

Influenza virus is a genome-based virus belonging to the orthomyxoviridae family, and when infected, causes severe respiratory distress.

Influenza viruses are divided into types A, B, and C, of which A and B are frequently infected with humans. In particular, serotypes of type A viruses are determined by differences in the amino acid sequence of hemagglutinin and neuraminidase proteins. Hemagglutinin (H) of influenza A virus has 16 different subtypes and neuraminidase enzyme (N) has 9 different subtypes. How is the combination of hemagglutinin protein and neuraminidase protein subtypes made? The new variant is determined. In humans, the hemagglutinin protein consists of three subtypes (H1, 2 and 3) and two neuraminidase proteins (N1 and 2), with the highest frequency of infection being H1N1 and H3N2. Influenza virus. Therefore, influenza virus vaccines also include H1N1 and H3N2 influenza viruses and type B influenza viruses of type A together.

In addition, since the influenza virus is an RNA virus with a high incidence of genotyping, even the same H1N1 virus may have low efficacy against the vaccine.

In addition, because vaccines are only effective against certain influenza viruses and have low immunity against influenza viruses of a variant, each year, a newly prepared vaccine must be vaccinated and the vaccine may be combined with other influenza viruses. This lowers the efficacy of the vaccine.

There are currently two drugs that inhibit the proliferation of influenza viruses. Janamivir and oseamimivir are used as therapeutic agents for influenza virus as drugs that inhibit the function of neuraminidase enzyme of influenza virus (Dreitlein WB., Maratos J., Brocavich. 2001, Clin Ther , 23: 327-355).

Ginamivir is sold under the trademark Relenza by Glaxowelcome Inc. and is inhaled to inhale medicine powder through the nose. The oseltamivir is sold under the name Tamiflu by Roche, and is used as an oral medication such as amantadine.

The rerenza may have trouble breathing and asthma is likely to worsen, and Tamiflu has side effects such as nausea and vomiting (McNicholl and McNicholl, 2001, Ann. Pharmacother. 35 (1): 57- 70) Efficacy varies greatly depending on the type of influenza virus.

In addition, ribavirin is a drug that inhibits the growth of influenza virus and is applied to both DNA and RNA virus infections (Magden J et al., 2005, Appl Microbiol Biotechnol., 66 (6): 612-621 ).

However, ribavirin also causes serious side effects such as anemia (Soriano V et al., 6, 2011, J Antimicrob Chemother, 66 (8): 1673-1686).

Corta virus (rota virus) is a genome of RNA that belongs to the family of coronaviridae, and when infected, causes respiratory disease or severe enteritis.

In addition, the corona virus is spread to the air or feces and has the characteristic that the onset of the disease varies depending on the animal. In humans, respiratory diseases such as SARS and cold are caused. In pigs and dogs, it causes severe enteritis, cats cause severe peritonitis, and rats cause hepatitis. Currently, there is no antiviral treatment for coronavirus infection, and there are no vaccines except for some swine coronaviruses, and the swine coronaviruses in which the vaccines are developed also have low vaccine efficacy due to the high frequency of viral variants.

In addition, rotavirus is a genome-based virus belonging to the genus Reoviridae, which causes severe enteritis.

Rotavirus is the most common cause of gastroenteritis in infants and is spread by feces or oral and causes symptoms such as vomiting, diarrhea, fever and abdominal pain. There is no antiviral treatment for rotavirus infection, and rotavirus vaccination is not included in national vaccination items. Rotavirus vaccines have live oral vaccines, but rotaviruses do not have significant preventive effects due to the presence of subgroups and various serotypes.

Citrus fruits are used for food as a fruit of the tree belonging to the genus citrus , and are used as juices to make juices and are sold as beverages. Among the fruits of citrus plants, citrus fruits are in high demand. However, because there are not many kinds of foods using citrus fruits, the industrial value of citrus fruits is stagnant, so it is necessary to create nutritional value for citrus fruits or create new value as favorite beverage and other functional foods.

Therefore, the present inventors showed low antiviral ability in the citrus juice, but the present invention was completed by confirming that the citrus juice was inoculated with Bacillus bacterium and fermented to have an excellent antiviral activity against influenza virus. It was.

An object of the present invention is to provide a pharmaceutical composition for the prevention and treatment of viral diseases containing a fermented product inoculated with Bacillus bacteria in a citrus fruit (Citrus) fruit grinding liquid as an active ingredient.

Another object of the present invention is to provide a fermented product fermented by inoculating Bacillus bacteria to bilberry fruit pulverized liquid as a health food for improving health against viral infectious diseases.

In addition, another object of the present invention to provide a feed additive for the prevention and improvement of livestock virus infectious diseases containing a fermented product inoculated and fermented Bacillus bacteria in the bilberry fruit juice.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of viral diseases containing a fermented product inoculated and fermented by Bacillus bacteria in pulverine fruit juice (crushed).

In addition, the present invention provides a health food for improving the health of the virus-infected diseases fermented by inoculating Bacillus bacteria in the citrus fruit juice.

In addition, the present invention provides a feed additive for the prevention and improvement of livestock virus infectious diseases containing a fermented product inoculated with Bacillus bacteria in a citrus fruit pulverized liquid as an active ingredient.

Fermented product of the genus citrus fruit pulverized liquid (grind) of the present invention with Bacillus bacterium has no human toxicity and exhibits a proliferation inhibitory effect against influenza virus, and thus, is a pharmaceutical composition for antiviral and health food. And it can be usefully used as an active ingredient of feed additives.

In addition, the fermented product of the genus citrus fruit grinding liquid of the present invention fermented with Bacillus bacteria shows an inhibitory effect on the growth of corona virus, as an active ingredient of antiviral pharmaceutical composition, health food and feed additives. It can be usefully used.

In addition, the fermented product fermented with the genus citrus fruit pulverized liquid of the present invention Bacillus bacteria exhibits a proliferation inhibitory effect on rotavirus, as an active ingredient of antiviral pharmaceutical composition, health food and feed additives It may be useful.

1 is a diagram confirming the antiviral ability against the influenza A virus (H1N1) of the fermented product inoculated with Bacillus bacteria (KJ000007) in the citrus fruit juice.

Figure 2 is a diagram confirming the antiviral ability against the influenza A virus (A / WS / 33) of the fermentation of fermented by inoculating Bacillus bacteria (KJ000007) in the citrus fruit juice.

Figure 3 is a diagram confirming the antiviral ability against influenza B virus fermented by inoculating Bacillus bacteria (KJ000007) in the citrus fruit juice.

4 is a diagram comparing the antiviral ability of the fermented corona virus inoculated with Bacillus bacteria (KJ0000007) in the citrus fruit pulverized liquid by the concentration of the antiviral ability of ribavirin and fruit pulverized liquid.

5 is a diagram showing the maximum antiviral activity against the corona virus of citrus fermentation products (fermentation product fermented by inoculating Bacillus bacteria (KJ000007) in citrus fruit juice) and ribavirin, citrus fruit juice.

Figure 6 is a diagram confirming the antiviral ability against human rotavirus fermented by inoculating Bacillus bacteria in bilberry fruit juice.

Figure 7 is a diagram confirming the antiviral activity against the pig rotavirus fermented by inoculating Bacillus bacteria in bilberry fruit juice.

Figure 8 is a diagram confirming the antiviral ability against bovine rotavirus fermented by inoculating Bacillus bacteria in bilberry fruit juice.

The present invention provides a pharmaceutical composition for the prevention and treatment of viral diseases containing a fermented product inoculated with Bacillus bacteria in a citrus fruit pulverized liquid (grind) as an active ingredient.

The citrus fruit is most preferably selected as a citrus fruit of the citrus tree, but not limited thereto, and any fruit belonging to the citrus tree may be used.

In the production of the fermented product, in the preferred embodiment of the present invention was prepared using the US Center for Biotechnology Information (USA, Accession No. (KJ000007), but Bacillus substilis bacteria if limited to this I never do that.

The viral diseases include, but are not limited to, diseases infected by influenza virus, diseases infected by corona virus, and diseases infected by rotavirus.

In a specific embodiment of the present invention, the inventors have produced bilberry fruit fermentation.

In addition, the present inventors confirmed the antiviral activity of the citrus fruit fermentation using influenza A virus (N1H1 type) and influenza B virus, the antiviral activity was significantly increased in the fermented product than the citrus juice, the fermentation product It showed antiviral activity comparable to Tamiflu used as a positive control (see Fig. 1).

In addition, the present inventors confirmed the antiviral activity of the citrus fruit fermentation using corona virus, the antiviral activity was significantly increased in the fermented product than the citrus juice, the fermented product is comparable to ribavirin used as a positive control group It showed antiviral activity (see FIG. 4).

In addition, the present inventors confirmed the antiviral activity of citrus fruit fermentation using human rotavirus, porcine rotavirus, and bovine rotavirus. As a result, the antiviral activity was significantly increased in the fermented product than the citrus juice. Antiviral activity was comparable to that of ribavirin used as a positive control (see FIG. 7).

In addition, it was confirmed that there is no toxicity even when administered to the mouse bilberry fruit fermentation of the present invention.

Therefore, the citrus fruit fermentation product of the present invention has no toxicity and is safe for the human body, and has an antiviral effect because it has a proliferation inhibitory effect against influenza virus, a proliferation inhibitory effect against coronavirus, and a proliferation inhibitory effect against rotavirus. It has been confirmed that it can be usefully used as a pharmaceutical composition.

The composition containing (including) the citrus fruit fermentation product of the present invention may further contain one or more active ingredients exhibiting the same or similar functions in addition to the above components.

As an active ingredient of the composition of the present invention, a pharmaceutically acceptable salt thereof may be added to the citrus fruit fermentation, and the salt may be an acid addition salt formed by a pharmaceutically acceptable free acid. useful.

The active ingredient of the composition can form a pharmaceutically acceptable acid addition salt according to a conventional method in the art.

The inorganic acid and organic acid may be used as the free acid, and hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, etc. may be used as the inorganic acid, and citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, Fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galluxuronic acid, embonic acid, glutamic acid or aspartic acid May be used, but is not limited thereto.

The composition of the present invention may further comprise a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose , Mannitol, malt, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, opiodry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate , Sucrose, dextrose, sorbitol, talc and the like can be used.

The pharmaceutically acceptable additive according to the present invention is preferably included 0.1 to 90 parts by weight (%) based on the total weight (%) of the composition, but is not limited thereto.

That is, the composition of the present invention can be administered in various oral and parenteral formulations during actual clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used It can be prepared using.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, It may be prepared by mixing sucrose, lactose or gelatin.

In addition to simple excipients, lubricants such as magnesium styrate talc may also be used.

Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents, water and liquid paraffin. .

Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used.

As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The composition of the present invention can be administered orally or parenterally according to the desired method, external or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection during parenteral administration It is desirable to choose an injection method.

Dosage varies depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and severity of disease.

Dosage units may contain, for example, one, two, three or four times the individual dosage, or they may contain 1/2, 1/3 or 1/4 times.

Individual dosages specifically contain amounts in which the effective drug is administered at one time, which typically corresponds to all, 1/2, 1/3 or 1/4 times the daily dose.

The effective dose of the composition of the present invention may be 0.0001 to 10 g / kg, specifically 0.001 to 1 g / kg, it may be administered 1 to 6 times a day. However, the dosage may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention in any way.

In addition, the present invention provides a health food for the prevention and improvement of viral diseases containing bilberry fruit fermentation as an active ingredient.

The citrus fruit is most preferably selected as a citrus fruit, but not limited to this, any fruit belonging to the citrus can be used.

The fermented product was prepared using Bacillus bacteria ( Bacillus substilis, NCBI Accession No. KJ000007) in citrus fruits, but is preferably prepared by inoculation of Bacillus bacteria ( Bacillus substilis ) in citrus fruits, but is not limited thereto. In addition, in the production of the fermentation, in the preferred embodiment of the present invention was prepared using the US Biotechnology Information Center (NCBI) Accession No. KJ000007, but not limited to Bacillus supstilis .

The viral disease includes, for example, a disease infected by at least one influenza virus selected from the group consisting of influenza viruses, a disease infected by corona virus, and a disease infected by rotavirus. One is not limited to this.

The health food of the present invention may be added with the bilberry fruit fermentation as it is, or may be used with other food or food ingredients, and may be appropriately used according to a conventional method.

There is no particular limitation on the kind of the health food.

Examples of foods to which the citrus fruit fermentation may be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, beverages, Tea, drink, alcoholic beverages and vitamin complexes, etc., and includes all of the health food in the usual sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates, etc. as additional components, as in the general beverage.

The above-mentioned natural carbohydrates are glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol and erythritol.

As the sweetening agent, natural sweetening agents such as taumartin, stevia grinding liquid, synthetic sweetening agents such as saccharin, aspartame, and the like can be used.

The ratio of the natural carbohydrate is generally about 0.01 to 0.04 weight (g), preferably about 0.02 to 0.03 weight (g) per 100 weight (g) of the composition of the present invention.

In addition to the above, the health food of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol And carbonation agents used in carbonated beverages.

Others may contain pulp for the production of natural fruit juices, fruit juice drinks and vegetable drinks.

These components can be used independently or in combination.

The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 weight (%) per 100 weight (%) of the composition of the present invention.

The present invention also provides a feed additive containing bilberry fruit fermentation as an active ingredient.

The citrus fruit is most preferably selected from the group consisting of citrus fruits, not limited to this, any fruit belonging to the citrus can be used.

In the production of the fermented product, in the preferred embodiment of the present invention was prepared using Bacillus bacteria, the US Biotechnology Information Center (NCBI) accession number (KJ000007), but not limited to Bacillus bacteria ( Bacillus substilis ).

The viral disease includes, for example, a disease infected by one or more viruses selected from the group consisting of influenza viruses, a disease infected by a corona virus, and a disease infected by a rotavirus. It is not limited to this.

Citrus fruit fermentation of the present invention is composed of 0.01 to 1% by weight relative to 100% (%) of the feed to be added, preferably citrus fruit fermentation is composed of 0.05 to 0.5% by weight (%) Most preferably, the bilberry fruit fermentation is 0.12 weight (%).

In addition, the feed additive may additionally contain a carrier that is acceptable to a unit animal.

In the present invention, the feed additive may be added as it is, or a known carrier, stabilizer, or the like, and various nutrients such as vitamins, amino acids, minerals, antioxidants and other additives may be added, if necessary. It may be in a suitable state such as powder, granules, pellets, suspensions and the like.

In the case of supplying the feed additive of the present invention, the unit animal may be supplied alone or mixed with feed.

Hereinafter, the present invention will be described in detail by the following Examples and Preparation Examples.

However, the following Examples and Preparation Examples specifically illustrate the present invention, and the contents of the present invention are not limited by the following Examples and Preparation Examples.

Reference Example 1: Control Drugs and Viruses

Tamiflu and ribovarin, test compounds, were purchased from Roche and Sigma, respectively.

The virus used in the present invention includes two types of influenza type A virus H1N1 type (A / PR / 8/34, A / WS / 33) and influenza type B virus (B / Lee / 40). ), The virus activity was cultured and measured, and then stored at -70 ° C for use.

Reference Example 2: Test Controls and Viruses

Ribavirin, a test compound, was purchased from Sigma.

The corona virus used in the present invention was supplied with corona virus (PEDV, porcine epidemic diarrhea virus) from the Ministry of Agriculture, Forestry and Livestock Quarantine and was stored at -70 ° C after culturing the virus activity.

Reference Example 3: Test Controls and Viruses

Ribavirin, the test compound, It was purchased from Sigma.

The viruses used in the present invention were three types of rotaviruses, and human rotaviruses were distributed from the American Cell Line Bank (ATCC), and swine rotaviruses and bovine rotaviruses were distributed from the Ministry of Agriculture, Forestry and Livestock Quarantine Headquarters, and then cultured and measured. , Stored at -70 ℃ was used.

Example 1 Citrus genus citrus ) Production of Fruit Ferment

<1-1> Preparation of Citrus Fruit Ferment

In order to prepare the citrus fruit fermentation product to be used in the present invention, citrus fruits were first prepared.

Specifically, after the whole citrus was ground, it was vacuum dried to powder. The powder was dissolved in dimethylsulfoxide and used at a concentration of 100 mg / mL.

Bacillus substilus ( Bacillus substilus. ) Bacteria (US National Biotechnology Information Center Accession No. KJ000007) was inoculated and incubated in citrus pulverized liquid medium to prepare a citrus fermentation product. It was used by dissolving in sulfoxide at a concentration of 100 mg / mL.

Example 2.

<2--1> Measurement of Growth Inhibitory Effect on Influenza A Virus (H1N1 Type) from Citrus Fruit Ferment

The inhibitory effect on the influenza A virus of the bilberry fruit ferment prepared by the method of Example 1 was confirmed using MDCK cell line, which is a dog kidney cell-derived cell line.

Specifically, the method for measuring the virus growth inhibitory ability of the citrus fruit pulverized liquid and the fermented product is two types of influenza virus type A, H1N1 (A / PR / 8 / 34) and (A / WS / 33) were measured for the effect of inhibiting viral activity, respectively.

MDCK cells were incubated in each well of a 96-well microplate until the bottom was full of cells, then the existing culture was removed and each well was washed twice with phosphate buffer. In addition, influenza virus solution adjusted to the concentration of TCID25 was added to each of the wells, and the bilberry fruit fermentation product and existing anti-influenza virus agents Tamiflu (Roche) and ribavirin (Sigma) were respectively added to dimethyl sulfoxide. It was used by dissolving.

The control group (Tamiflu, ribavirin) was administered to each well at a final concentration of 0.1 ~ 100 ㎍ / ml, bilberry fruit fermentation was administered to each well at a final concentration of 1 ~ 1000 ㎍ / ml, 48 hours after incubation The infection experiment was terminated, 70% acetone was added to each well by 100 ul, then left at -20 ° C for 1 hour, and dried in a drier.

The dried product was stained for 30 minutes by adding 100 ul of 0.4% (w / v) SRB (sulforhodamine B) solution dissolved in 1% (v / v) acetic acid, and 1% (v / v) washed four times with acetic acid and dried. Again 100 ul of 10 mM Tris solution (pH 10.5) was added to the well plate to dissolve the dye bound to the cells and the absorbance was measured at 562 nm.

Using the results of the absorbance, it was calculated as shown in the following [Equation 1] to confirm the cell survival rate (%), wherein the virus-treated group (A), citrus fruit pulverized liquid and fermented product of the present invention The treated group (B), the virus-treated group (C) and the virus and the citrus fruit pulverized stock solution and the fermented product of the present invention (D).

In addition, it was calculated by the following Equation 2 to determine the virus growth inhibition capacity (%) value.

The statistics for the results are expressed as the mean value and the standard deviation of the values of three experiments under the same conditions.

[Equation 1]

[Equation 2]

As a result, as shown in FIG. 1, the maximum antiviral activity of Tamiflu against influenza A virus (H1N1 type) was 79.70 ± 4.63% at a concentration of 100 μg / ml, and the maximum antiviral activity of citrus juice was 1000. 12.98 ± 12.55% at the concentration of μg / ml, respectively. On the other hand, the maximum antiviral activity of citrus ferment was 50.42 ± 11.15% at the concentration of 1000 ㎍ / ml.

Therefore, fermented products inoculated with Bacillus subtilis ( Bacillus substilus. ) (US National Biotechnology Information Center Accession No. KJ000007) in the pulverized stock of citrus fruits increased four times the antiviral activity.

In addition, the maximum antiviral activity of Tamiflu against another influenza A virus (A / WS / 33) is 19.08 ± 14.35% at a concentration of 100 μg / ml and ribavirin is 100 μg / ml as shown in FIG. 2. The concentration was 68.74 ± 8.48%. The maximum antiviral activity of the citrus fruits was 0.58 ± 10.32% at a concentration of 1000 μg / ml. On the other hand, the fermented products inoculated with Bacillus substilus. .

As such, the citrus fermented product showed much higher antiviral activity than the ground liquid.

<2--2> Measurement of the Inhibitory Effect of Citrus Fruit Ferment against Influenza B Virus.

In order to measure the effect of inhibiting proliferation against influenza B virus of the citrus fruit pulverized liquid and the fermented product prepared by the method of [Example 1], the method of [Example 2] was performed.

As a result, as shown in FIG. 3, the maximum antiviral activity of Tamiflu against influenza B virus was 1.22 ± 13.13% at a concentration of 100 μg / ml, and ribavirin at 52.36 ± 5.82% at a concentration of 100 μg / ml. It was. The maximum antiviral activity of citrus pulverized liquid was 9.53 ± 1.40% in the concentration range of 1 ~ 1000 ㎍ / ml. On the other hand, the maximum antiviral activity of citrus ferment was 46.22 ± 14.96% at the concentration of 1000 ㎍ / ml.

In the results, it was confirmed that the citrus fermented product increased antiviral activity superior to citrus pulverized liquid (FIG. 3).

Example 3 Measurement of the Effect of Growth Inhibition on Corona Virus of Tangerine Fruit Ferment

The proliferation inhibitory effect on the corona virus of the bilberry fruit fermentation prepared by the method of Example 1 was confirmed using a Vero cell line, a monkey kidney cell-derived cell line.

Specifically, the method for measuring the virus growth inhibition ability of the citrus fruit pulverized liquid and the fermented product was measured by the virus activity inhibitory effect on the human corona virus purchased from ATCC according to the method of (KR) 682069.

MA104 cells were incubated in each well of a 96-well microplate until the bottom was full of cells, then the existing culture was removed and each well was washed twice with phosphate buffer. In addition, an influenza virus solution adjusted to the concentration of TCID25 was added to each of the wells, and the bilberry fruit fermentation product and the existing antiviral agent ribavirin (Sigma) were dissolved in dimethyl sulfoxide, respectively.

The control (ribavirin) was administered to each well at a final concentration of 0.1 ~ 100 ㎍ / ml, bilberry fruit fermentation was administered to each well at a final concentration of 1 ~ 1000 ㎍ / ml, 48 hours after the culture After the addition of 100 ul of 70% acetone to each well, it was left at -20 ℃ for 1 hour, and dried in a dryer.

The dried product was stained for 30 minutes by adding 100 ul of 0.4% (w / v) SRB (sulforhodamine B) solution dissolved in 1% (v / v) acetic acid, and 1% (v / v) washed four times with acetic acid and dried.

Again 100 ul of 10 mM Tris solution (pH 10.5) was added to the well plate to dissolve the dye bound to the cells and the absorbance was measured at 562 nm.

Using the results of the absorbance, it was calculated as shown in the following [Equation 1] to confirm the cell survival rate (%), wherein the virus-treated group (A), citrus fruit pulverized liquid and fermented product of the present invention The treated group (B), the virus-treated group (C) and the virus and the citrus fruit pulverized stock solution and the fermented product of the present invention (D). In addition, it was calculated by the following Equation 2 to determine the virus growth inhibition capacity (%) value.

The statistics for the results are expressed as the mean value and the standard deviation of the values of three experiments under the same conditions.

[Equation 1]

[Equation 2]

As a result, as shown in FIG. 4, the maximum antiviral activity of ribavirin against coronavirus was 66.56 ± 9.44% at a concentration of 100 μg / ml, and the maximum antiviral activity of citrus pulverized solution was 1000 μg / ml. At 0.16 ± 4.30% respectively. In contrast, the maximum antiviral activity of citrus ferment was 100.22 ± 38.56% at a concentration of 1000 μg / ml.

Therefore, fermented products inoculated with Bacillus substilus ( Bacillus substilus. ) (US National Biotechnology Information Center Accession No. KJ000007) in the pulverized stock of bilberry fruit increased 10 times more antiviral activity than the pulverized juice.

From the above results, it can be seen that the citrus fermentation composition according to the present invention exhibits excellent antiviral activity against coronaviruses, and has a very high utility in preventing and treating coronavirus infections in industries such as medicine, food, and feed.

Example 4.

<4-1> Determination of Growth Inhibitory Effects of Human Rotavirus from Tangerine Fruit Ferment

The inhibitory effect on the influenza A virus of the bilberry fruit ferment prepared by the method of Example 1 was confirmed using a MA104 cell line, which is a monkey kidney cell-derived cell line.

Specifically, the method for measuring the virus growth inhibition ability of the citrus fruit pulverized liquid and the fermented product measured the virus activity inhibitory effect on the human rotavirus purchased from ATCC according to the method of (KR) 662069.

MA104 cells were incubated in each well of a 96-well microplate until the bottom was full of cells, then the existing culture was removed and each well was washed twice with phosphate buffer.

In addition, an influenza virus solution adjusted to the concentration of TCID25 was added to each of the wells, and the bilberry fruit fermentation product and the existing antiviral agent ribavirin (Sigma) were dissolved in dimethyl sulfoxide, respectively.

The control (ribavirin) was administered to each well at a final concentration of 0.1 ~ 100 ㎍ / ml, bilberry fruit fermentation was administered to each well at a final concentration of 1 ~ 1000 ㎍ / mL, 48 hours after the infection test After the addition of 100ul of 70% acetone to each well, it was left at -20 ℃ for 1 hour, and dried in a dryer.

The dried product was stained for 30 minutes by adding 100 ul of 0.4% (w / v) SRB (sulforhodamine B) solution dissolved in 1% (v / v) acetic acid, and 1% (v / v) washed four times with acetic acid and dried.

Again 100 ul of 10 mM Tris solution (pH 10.5) was added to the well plate to dissolve the dye bound to the cells and the absorbance was measured at 562 nm.

Using the results of the absorbance, it was calculated as shown in the following [Equation 1] to confirm the cell survival rate (%), wherein the virus-treated group (A), citrus fruit pulverized liquid and fermented product of the present invention The treated group (B), the virus-treated group (C) and the virus and the citrus fruit pulverized stock solution and the fermented product of the present invention (D).

In addition, it was calculated by the following Equation 2 to determine the virus growth inhibition capacity (%) value.

The statistics for the results are expressed as the mean value and the standard deviation of the values of three experiments under the same conditions.

[Equation 1]

[Equation 2]

As a result, as shown in FIG. 6, the maximum antiviral activity of ribavirin against human rotavirus was 50.44 ± 6.88% at a concentration of 100 μg / ml, and the maximum antiviral activity of citrus juice was 1000 μg / ml. The concentrations were 7.86 66.81% respectively. On the other hand, the maximum antiviral activity of citrus ferment was 72.60 ± 4.67% at the concentration of 1000µg / ml.

Thus, fermented products inoculated with Bacillus substilus ( Bacillus substilus. ) Bacteria (US National Biotechnology Information Center Accession No. KJ000007) in the pulverized stock of citrus fruits confirmed that the antiviral activity was increased by 9 times or more. Can be.

<4-2> Determination of Growth Inhibitory Effects of Pork Rotavirus from Tangerine Fruit Ferment

In order to measure the effect of inhibiting proliferation against the influenza B virus of the tangerine fruit pulverized liquid prepared by the method of Example 1 and the fermented product, it was carried out by the <4-1> method of Example 4.

Specifically, the method for measuring the virus growth inhibition ability of the citrus fruit pulverized liquid and the fermented product measured the virus activity inhibitory effect on the swine rotavirus distributed by the Agriculture, Forestry and Livestock Quarantine Headquarters according to the method of (KR) Patent No. 682069.

As a result, as shown in FIG. 7, ribavirin to porcine rotavirus showed 50.44 ± 6.88% at a concentration of 100 μg / ml. The maximum antiviral activity of citrus juice It showed 7.25 ± 2.14% in the concentration range of 1 ~ 1000㎍ / ml. On the other hand, the maximum antiviral activity of citrus ferment was 61.30 ± 2.17% at the concentration of 1000㎍ / ml.

In the above results, it can be seen that the citrus fermented product increased the antiviral activity by 8 times or more than the citrus juice.

<4-3> Determination of Growth Inhibitory Effect on Bovine Rotavirus of Tangerine Fruit Ferment

In order to measure the growth inhibitory effect on the bovine rotavirus and the citrus fruit pulverized liquid prepared by the method of Example 1, it was carried out by the <4-1> method of Example 4.

Specifically, the method for measuring the virus growth inhibition ability of the citrus fruit pulverized liquid and the fermented product was measured the effect of inhibiting the virus activity against bovine rota virus distributed by the Ministry of Agriculture, Forestry and Livestock Quarantine based on the method of (KR) Patent No. 682069.

As a result, as shown in FIG. 8, ribavirin against bovine rotavirus showed 71.50 ± 0.88% at a concentration of 100 μg / ml. The maximum antiviral activity of citrus pulverized liquid was 27.33 ± 9.61% in the concentration range of 1-1000 ㎍ / ml. On the other hand, the maximum antiviral activity of citrus ferment was 64.18 ± 6.37% at the concentration of 1000µg / ml.

In the above results, it can be seen that the citrus fermented product increased the antiviral activity by 2 times or more than the citrus crushed liquid (FIG. 8).

From the above results, it can be seen that the citrus fermentation composition according to the present invention exhibits excellent antiviral activity against rotavirus.

Example 5. Toxicity Confirmation of Citrus Fruit Ferment

An experiment was conducted to confirm the toxicity of the bilberry fruit ferment prepared by the method of [Example 1].

Specifically, after dissolving the bilberry fruit fermentation in water, it was observed for 7 days after the administration of 10 g / kg to each of the mice (10 per group), as a result there was no rat death. As a result, the bilberry fruit fermentation of the present invention was proved to be non-toxic.

Preparation Example 1 Preparation of Pharmaceutical Formulation

<1-1> Preparation of Powder

2 g of bilberry fruit fermentation of the present invention

1 g lactose

The above ingredients were mixed and filled in airtight cloth to prepare a powder.

<1-2> Preparation of Tablet

100 mg of bilberry fruit fermentation of the present invention

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

<1-3> Preparation of Capsule

100 mg of bilberry fruit fermentation of the present invention

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

<1-4> Preparation of the ring

1 g of bilberry fruit fermentation of the present invention

Lactose 1.5 g

1 g of glycerin

Xylitol 0.5 g

After mixing the above components, it was prepared to be 4 g per ring in a conventional manner.

<1-5> Preparation of Granules

150 mg of bilberry fruit fermentation of the present invention

Soy grinding powder 50 mg

Glucose 200 mg

Starch 600 mg

After mixing the above components, 100 mg of 30% ethanol was added and dried at 60 ° C. to form granules, which were then filled into fabrics.

Preparation Example 2 Preparation of Health Food

<2-1> Preparation of Flour Food

0.5 to 5.0 weight percent of the bilberry fruit fermentation product of the present invention was added to the flour and the mixture was used to prepare bread, cake, cookies, crackers and noodles.

<2-2> Preparation of Soups and Gravys

0.1-5.0 weight (%) of the citrus fruit fermentation product of the present invention was added to the soup and the juice, based on the total weight (%), to prepare the health-producing meat products, the soup of the noodles, and the juice.

<2-3> Preparation of Ground Beef

Health weight ground beef was prepared by adding 10 weight% (%) of the bilberry fruit fermentation of the present invention to the ground weight (%).

<2-4> Production of Dairy Products

To the total weight (%) was added 5-10 weight (%) of the bilberry fruit fermentation of the present invention to milk, using the milk to prepare a variety of dairy products such as butter and ice cream.

<2-5> Preparation of Wire

Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted to prepare a powder having a particle size of 60 mesh.

Black beans, black sesame seeds, and perilla were also steamed and dried by a known method, and then ground to a powder having a particle size of 60 mesh.

The tangerine fruit fermentation product of the present invention was concentrated under reduced pressure in a vacuum concentrator, and the dried product obtained by drying with a spray and a hot air dryer was pulverized to a particle size of 60 mesh by a grinder to obtain a dry powder.

The grains, seeds and the bilberry fruit fermentation of the present invention prepared in the above were formulated in the following ratio.

Cereals (30 g of brown rice, 15 g of barley, 20 g of barley),

Seeds (7 weight (g) perilla, 8 weight (g) black bean, 7 weight (g) black sesame),

Tangerine fruit fermentation product (3 weight (g)) of the present invention,

Ganoderma lucidum (0.5 g (g)),

Foxglove (0.5 weight (g))

Preparation Example 3 Preparation of Beverage

<3-1> Preparation of health drink

Instantly mix homogeneous ingredients such as liquid fructose (0.5 g), oligosaccharide (2 g), sugar (2 g), salt (0.5 g), water (75 g) and 5 g of the citrus fruit fermentation product of the present invention After sterilization, it was prepared by packing it in a small packaging container such as a glass bottle or a plastic bottle.

<3-2> Preparation of Vegetable Juice

5 g of the bilberry fruit fermentation of the present invention was added to 1,000 ml of tomato or carrot juice to prepare vegetable juice.

<3-3> Preparation of Fruit Juice

1 g of the bilberry fruit fermentation of the present invention was added to 1,000 ml of apple or grape juice to prepare a fruit juice.

Preparation Example 4 Preparation of Feed

<4-1> Preparation of Feed Additives

Citrus fruit ferment 0.1 ~ 1 weight (%) of the present invention

Tricalcium phosphate 1 ~ 20 weight (%)

Vitamin E 0.01-0.1 weight (%)

Enzyme powder 1 ~ 10 weight (%)

Lactobacillus 0.1-10 weight (%)

Glucose 58.9 ~ 90 Weight (%)

<4-2> Preparation of Feed

Using the feed additive of the manufacturing method <4-1> as an active ingredient to prepare a feed with the following composition.

<4-1> 0.1 to 10 weight% of feed additives

Bran 40-49.9 Weight (%)

Milo 21.20 Weight (%)

Soybean Meal 20.00 Weight (%)

Fish flour 3.00 weight (%)

Molasses 4.00 weight (%)

Mineral 1.53 weight (%)

0.27 weight (%) of vitamin

Fermented product obtained by fermenting genus citrus fruit pulverized liquid (grind) with Bacillus bacterium has no human toxicity and shows a proliferation inhibitory effect against influenza virus, corona virus and rotavirus, In addition, it may be widely used as an active ingredient in health food and feed additives.

Claims (16)

1. In the composition for antiviral,

   Genus citrus ( genus citrus ) Antiviral composition comprising a citrus fruit fermentation, characterized in that it contains as an active ingredient fruit fermentation.
2. The method of claim 1,

   The citrus fruit is an antiviral composition comprising a citrus fruit fermented product as an active ingredient, characterized in that at least one fruit selected from the group of citrus fruit.
3. The method of claim 1,

   The fermentation product of the citrus fruit, the antiviral composition comprising a citrus fruit fermentation product, characterized in that the fermentation of the juice of the fruit of the tangerine ( Bacillus sp. ) Strain.
4. The method of claim 3, wherein

   The Bacillus strain is an antiviral composition comprising a bilberry fruit fermentation, characterized in that the US National Biotechnology Information Center Accession No. KJ000007.
5. The method according to any one of claims 1 to 4,

   The genus citrus fruit ( genus citrus ) fruit fermentation antiviral composition comprising a bilberry fruit fermentation, characterized in that it is used in the pharmaceutical composition for the prevention and treatment of viral diseases.
6. The method of claim 5,

   The viral disease is an antiviral composition comprising a bilberry fruit fermentation, characterized in that the disease is infected by any one or more viruses selected from the group consisting of influenza virus as an active ingredient.
7. The method of claim 5,

   The viral disease is a composition for antiviral comprising a bilberry fruit fermentation, characterized in that the disease is infected by any one or more viruses selected from the group consisting of rotavirus.
8. The method of claim 5,

   The viral disease is a composition for antiviral comprising a bilberry fruit fermentation, characterized in that the disease is infected by a corona virus as an active ingredient.
9. The method according to any one of claims 1 to 4,

   The genus citrus fruit ( genus citrus ) fruit is an anti-viral composition comprising a citrus fruit fermentation as an active ingredient, characterized in that it is used in the health food for the prevention and improvement of viral diseases.
10. The method of claim 9,

    The viral disease is an antiviral composition comprising a bilberry fruit fermentation, characterized in that the disease is infected by any one or more viruses selected from the group consisting of influenza virus as an active ingredient.
11. The method of claim 9,

    The viral disease is an antiviral composition comprising a bilberry fruit fermentation, characterized in that the disease is infected by any one or more viruses selected from the group consisting of rotavirus as an active ingredient.
12. The method of claim 9,

    The viral disease is a composition for antiviral comprising a bilberry fruit fermentation, characterized in that the disease is infected by a corona virus as an active ingredient.
13. The method according to any one of claims 1 to 4,

    The genus citrus fruit ( genus citrus ) fruit fermentation antiviral composition comprising a bilberry fruit fermentation, characterized in that used as a feed additive for the prevention and improvement of viral diseases.
14. The method of claim 13,

    The viral disease is an antiviral composition comprising a bilberry fruit fermentation, characterized in that the disease is infected by any one or more viruses selected from the group consisting of influenza virus as an active ingredient.
15. The method of claim 13,

    The viral disease is a composition for antiviral comprising a bilberry fruit fermentation, characterized in that the disease is infected by any one or more viruses selected from the group consisting of rotavirus.
16. The method of claim 13,

    The viral disease is a composition for antiviral comprising a bilberry fruit fermentation, characterized in that the disease is infected by a corona virus as an active ingredient.

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