KR102402794B1 - Composition for antiviral activity containing oil distilled from Pinus densiflora xylem - Google Patents

Abstract

The present invention relates to an antiviral composition containing Pinus densiflora xylem oil as an active ingredient. The Pinus densiflora xylem (trunk and stem) oil extracted by steam distillation has an excellent antiviral effect against influenza A virus and coronavirus. Specifically, the Pinus densiflora xylem oil inhibits the activity and proliferation of influenza A virus (H1N1) and inhibits the activity and proliferation of feline coronavirus (FCoV). Therefore, the composition containing Pinus densiflora xylem oil as an active ingredient can be usefully used in antiviral functional cosmetics, multi-purpose detergents, preservatives, additives, foods, pharmaceuticals, and the like.

Description

An antiviral composition containing oil extracted from pine xylem as an active ingredient {Composition for antiviral activity containing oil distilled from Pinus densiflora xylem}

The present invention relates to an antiviral composition comprising pine ( Pinus densiflora Siebold & Zucc.) wood oil as an active ingredient. Pine xylem (trunk and stem) oil extracted by steam distillation has excellent antiviral effect against influenza A virus and coronavirus.

Specifically, pine wood oil inhibits the activity and proliferation of influenza A virus (H1N1), and inhibits the activity and proliferation of Feline coronavirus (FCoV). Therefore, the composition containing pine xylem oil as an active ingredient can be usefully used in antiviral functional cosmetics, multi-purpose cleaners, preservatives, additives, foods, pharmaceuticals, and the like.

As a result of the present invention, the task information is as follows.

1. Research project name: Nature life industry technology development project

2. Research project name: Development of commercial products using Gyeongbuk Patent Pine Rectification

3. Name of project performing organization: National Baekdudaegan Arboretum

4. Research period: 2021.04.01 ~ 2021.12.31

Influenza virus is a single-stranded RNA virus, classified into three types A, B, and C according to the antigenicity of nucleoprotein (NP) and matrix antigen, and 16 types according to the type of hemagglutinin (HA), a glycoprotein protruding from the virus envelope is divided into subtypes (H1-H16), and again divided into nine subtypes (N1-N9) according to the type of neuraminidase. Hemagglutinin, as the most important antigenic determinant, binds to the sialic acid receptor on the surface of the host cell and facilitates the penetration of the virus into the host. Neuraminidase catalyzes the separation of virions by removing sialic acid from the host cell, and prevents virion aggregation, allowing the newly created virus to be easily released. In addition, the M2 protein, which is present in a small amount in the viral membrane protein, acts as an ion channel to control the pH inside the virus, thereby exposing the RNA of the virus into the host cell.

Among them, influenza A is a zoonotic disease that infects animals and humans and has a strong contagiousness, causing a worldwide pandemic, causing many casualties in human history. The Spanish flu, which was highly contagious from 1918 to 1920 and swept through Europe, killed about 40 million people, making it the deadliest flu in history.

In March 2009, the novel influenza A virus H1N1 spread worldwide, resulting in over 10,000 deaths. In Korea, more than 860,000 people were confirmed and 270 deaths occurred. In addition, according to a study by the Korea Institute of Health and Medical Sciences, it is estimated that 2,370 people die annually due to seasonal influenza.

Meanwhile, coronavirus is one of the three major viruses that cause colds in humans along with adenovirus and rhinovirus, and is an RNA virus with a gene size of 27 to 32 kb that can infect humans and various animals. When viewed with an electron microscope, the surface of the virus particle protrudes like a protrusion, and this shape is reminiscent of a crown or the corona of the sun.

It accounts for 10 to 30% of adult colds that occur mainly in the cold winter, and the main symptom is a nasal cold accompanied by a headache, sore throat, or cough. Coronaviruses were first discovered in chickens in the 1930s and then in animals such as dogs, pigs, and birds, and in humans in the 1960s. Coronaviruses can infect both animals and humans, and as the human activity area expands, viruses that only spread among animals cause genetic mutations to survive and pass to humans. Examples include SARS (bats and civets), MERS (bats and camels), and COVID-19 (bats). (See Fig. 6)

On the other hand, the present invention relates to the antiviral function of pine xylem oil, and when reviewing the prior art literature, Korean Patent Publication No. 10-2021-0133861 discloses a composition for preventing swine influenza infection using pine pine pine pine extract. . Referring to claim 6, it is described that pine resin is extracted with methanol or ethanol and fractionated with ethyl acetate.

In addition, Korean Patent No. 10-0743861 discloses 'a composition for the treatment and prevention of human diseases caused by viruses containing pine needles extract'. Referring to claim 1, it is described that ethanol, dichloromethane or ethyl acetate soluble extract of pine needles is contained as an active ingredient.

Republic of Korea Patent Publication No. 10-2021-0133861 (published on January 8, 2021) Republic of Korea Patent Publication No. 10-0743861 (Registered on July 24, 2007)

An object to be solved is to provide an antiviral composition comprising, as an active ingredient, an oil obtained by distilling and extracting pine xylem by steam distillation.

The solution is an antiviral composition comprising pine xylem oil obtained by steam distillation of pine ( Pinus densiflora Siebold & Zucc.) xylem as an active ingredient.

In the above, the pine wood oil is characterized in that it inhibits the activity and proliferation of influenza A virus (H1N1), and inhibits the activity and proliferation of Feline coronavirus (FCoV).

In the above, pine xylem oil is included at a concentration of 10 ^-3 to 10 ^-2 v/v % for antiviral functionality against influenza A, and 10 ^-2 v for antiviral functionality against Feline coronavirus (FCoV) It is characterized in that /v % concentration is included.

The solution is an antiviral food composition containing pine xylem oil obtained by steam distillation of pine ( Pinus densiflora Siebold & Zucc.) xylem as an active ingredient.

The solution is an antiviral cosmetic composition containing pine xylem oil obtained by steam distillation of pine ( Pinus densiflora Siebold & Zucc.) xylem as an active ingredient.

The solution is an antiviral cleaner containing pine xylem oil obtained by steam distillation of pine ( Pinus densiflora Siebold & Zucc.) xylem as an active ingredient.

The present invention secures productivity and economy because all of the pine wood parts are utilized. According to the present invention, the pine xylem oil extracted by steam distillation has the effect of inhibiting the activity and proliferation of influenza A virus (H1N1), and inhibiting the activity and proliferation of Feline coronavirus (FCoV). Since the composition containing pine wood oil according to the present invention has antiviral function, it can be usefully used in antiviral functional cosmetics, multipurpose cleaners, preservatives, additives, foods, pharmaceuticals, and the like.

1 is a view describing the cytotoxicity results of pine xylem oil.
Figure 2 is a view describing the antiviral test results for influenza A type of pine xylem oil.
3 is a photograph of plaque assay results for influenza A of pine wood oil.
4 is a view describing the antiviral test results for FCoV of pine xylem oil.
Figure 5a is a photograph of the TCID50 test result for FCoV of the control group, and Figure 5b is a photograph of the TCID50 test result for FCoV of pine xylem oil.
6 is a view showing the classification of the corona virus.

The terms or words used in the present specification and claims are not to be construed as limited in their ordinary or dictionary meanings, and that the inventor may appropriately define the concepts of the terms in order to best describe his invention. Based on the principle, it should be interpreted as meaning and concept consistent with the technical idea of the present invention.

Therefore, the matters described in the embodiments, reference examples and drawings described in this specification are only the most preferred examples of the present invention and do not represent all of the technical spirit of the present invention, so they can be substituted at the time of the present application It should be understood that various equivalents and modifications may exist.

The pine tree ( Pinus densiflora Siebold & Zucc.) wood oil used in the following experimental examples is an oil obtained by extracting and collecting the trunk and stems of pine trees by steam distillation.

'Neck' is one of the components of the vascular bundle and refers to a complex tissue composed of ducts, trabeculae, xylem fibers, and xylem tissues. It serves as a passageway for water and nutrients and as a mechanical support for the tree.

The steam distillation method is a method of collecting volatile components by distilling through steam, and is a method of extracting a water immersion liquid formulation to repeatedly obtain a distillate.

Experimental Example 1. Influenza A antiviral test of pine xylem oil

1 is a view describing the cytotoxicity results of pine xylem oil. Figure 2 is a view describing the antiviral test results for influenza A type of pine xylem oil. 3 is a picture of plaque assay results for influenza A of pine wood oil.

[Cytotoxicity evaluation]

For the cytotoxicity evaluation method, Modified ISO 10993-5 was used, and CRFK cells were used. To determine whether pine xylem oil exerts cytotoxicity, cell viability was measured using a CRFK cell and a Thiazolyl Blue Tetrazolium Bromide (MTT) assay, a cell viability measurement technique.

It was dispensed in a 96-well plate at a concentration of 4×10 ⁴ cells/ml and cultured at 37° C., 5% CO ₂ in an incubator for 48 hours. The cultured cells were treated with pine xylem oil at different concentrations and reacted for 24 hours, then treated with 10 μl of MTT reagent at a concentration of 5 mg/ml and incubated for 4 hours. Then, the sample and MTT reagent were removed, and the active cells reduced MTT. To measure the formazan crystal produced by adding 100 μl of DMSO, the cell viability was measured using absorbance at 540 nm.

As a result of the experiment, as shown in FIG. 1, the cell viability of pine xylem oil was 10 ^-2 A cell viability of 81.7% or more was confirmed at a concentration of v/v % or less.

[Plaque assay of pine xylem oil]

Modified ASTM E1052-11 method was used to test the antiviral effect of pine wood oil against influenza A virus (H1N1), and MDCK cells were used as host cells.

Infectivity assays: The greatest characteristic of viruses is that they multiply within cells, and virus-infected cells undergo a series of biochemical and morphological changes and, in most cases, die. The morphological change in virus-infected cells is called cytopathic effect (CPE) and is expressed in the form of cell rounding, cell fusion (syncytia formation), and cell lysis.

A. Plaque assay: Plaque is a single-layered cell region that has undergone cellular degeneration due to virus infection. Normal, uninfected cells around the plaque are stained with vital dye, so they are distinguished. Just as a characteristic colony is created for each bacteria, a characteristic plaque with a different size and shape is created for each virus.

○ Under the theory that one infectious virus particle forms a single plaque, plaque counting has been applied to the quantification of various viruses, but plaque assay can only be used to quantify CPE-indicating viruses.

○ After preparing a cell monolayer, dilute the virus suspension to be quantified 10-fold from 10 ^-1 to 10 ^-6 .

○ Inoculate 0.1 mL to 0.5 mL of the diluted solution. After adsorbing the virus for 1-2 hours, overlay media is added.

○ Overlay media supplies nutrients and makes the media in a gel state to prevent the virus propagated in the first infected cells from spreading through the media and infecting surrounding cells.

○ Plaque appears as clear, colorless foci in contrast to the surrounding healthy cell monolayer that is not infected with the virus. To facilitate plaque counting, cell monolayers are sometimes stained with vital dyes such as neutral red or crystal violet.

○ Healthy cells absorb dye and become stained, and infected cells, that is, the plaque area, remain colorless without being stained. The titer of the virus stock is expressed in plaque forming units per milliliter (PFU/mL) after counting the number of plaques and taking into account the inoculum and dilution factor.

○ To minimize errors in calculation, multiple dilution multiples are used, and titer is calculated by counting the plates on which 20-100 plaques are formed.

Plaque number x dilution factor x inoculum converted to mL = ()PFU/mL

As a result of the experiment, as shown in FIGS. 2 and 3 , the pine xylem oil of the present invention showed a 99.67% reduction in virus proliferation level in influenza A (H1N1). This indicates that the pine xylem oil of the present invention has high antiviral activity in reducing influenza A virus.

Experimental Example 2. FCoV antiviral test of pine xylem oil

4 is a view describing the antiviral test results for FCoV of pine xylem oil. Figure 5a is a photograph of the TCID50 test result for FCoV of the control group, and Figure 5b is a photograph of the TCID50 test result for FCoV of pine xylem oil.

[TCID50 Test of Pine Wood Oil]

Modified ASTM E1052 method was used to test the antiviral effect of pine xylem oil against feline coronavirus (FCoV), and CRFK cells were used as host cells.

The inactivation test for coronavirus derived antiviral results through the tissue culture infectious dose (TCID50) method.

The 50% tissue culture infectious dose (TCID50) is a measure of infectious virus titer. This endpoint dilution assay quantifies the amount of virus required to kill 50% of infected hosts or produce a cytopathic effect (CPE) in 50% of inoculated cultured cells. This assay may be more common in the field of clinical research to determine lethal doses of virus or where the virus does not form plaques. By observing the cell monolayer inoculated with the virus for each 10-fold, the presence or absence of CPE is determined and the percentage of infected wells is calculated. In TCID50 assays, the time normally required to establish infection in cell culture is up to 7 days.

TCID50 analysis shows the titer of the virus dilution factor that infects 50% by inoculating 5 or more (usually 8-10 test units) animal cells for each multiple of the virus suspension diluted 10 fold. By observing the cell monolayer inoculated with the virus for each fold, the presence or absence of CPE is determined, and the percentage of infected wells is calculated. The 50% endpoint is calculated using the Reed-Muench method.

As a result of the experiment, as shown in FIGS. 4 and 5b , the pine xylem oil of the present invention showed a 68.38% reduction in virus proliferation level in the feline coronavirus (FCoV).

Example 1. Antiviral composition containing oil extracted from pine xylem as an active ingredient

The composition according to the present invention contains pine xylem oil obtained by extracting and collecting pine tree ( Pinus densiflora Siebold & Zucc.) xylem - trunk and stem - by steam distillation method as an active ingredient.

Based on the results of the above experimental examples, pine xylem oil inhibits the activity and proliferation of influenza A virus (H1N1), and inhibits the activity and proliferation of Feline coronavirus (FCoV). Therefore, the composition containing pine xylem oil as an active ingredient can be usefully used in antiviral functional cosmetics, multi-purpose cleaners, preservatives, additives, foods, pharmaceuticals, and the like.

Based on the results of the above experimental examples, preferably, the composition according to the present invention contains 10 ^-3 to 10 ^-2 v / v % concentration of pine wood oil for antiviral functionality against influenza A, and Feline coronavirus ( FCoV) contains pine xylem oil at a concentration of 10 ^-2 v/v % for antiviral functionality. Depending on the application used, the degree of dilution may vary.

The present invention may provide a formulation formulated in a pharmaceutical unit dosage form by including the pine xylem oil as an active ingredient and adding a pharmaceutically acceptable carrier, excipient or diluent. Here, the carrier, excipient, and diluent include toz, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, undecided. vaginal cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In addition, the pharmaceutical dosage form may be used in the form of a pharmaceutically acceptable salt, and may be used alone or in combination with other pharmaceutically active compounds as well as in an appropriate group. In addition, when formulating the active ingredient, it can be prepared using a commonly used diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, and a surfactant. In addition, the pharmaceutical dosage forms are each formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions according to conventional methods to be used. can

In the solid formulation for oral administration, at least one or more excipients, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc. may be mixed with the extract. It can be prepared by mixing. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used.

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

The preferred dosage of the composition of the present invention varies depending on the condition and weight of the patient, the degree of disease, age, sex, drug form, administration route and period, but may be appropriately selected by those skilled in the art. However, for a desirable effect, the composition of the present invention is preferably administered at 0.001 to 300 mg/kg, and the administration may be administered once a day, or may be administered in several divided doses. The above dosage does not limit the scope of the present invention in any way.

The composition of the present invention may be administered to mammals such as rats, mice, livestock, and humans by various routes. Any mode of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous injection.

It is possible to provide an antiviral functional food by adding a food supplement additive to the active ingredient of the present invention.

Foods to which the active ingredient can be added include, for example, various foods, beverages, gums, tea, vitamin complexes, health functional foods, and the like.

The amount of the active ingredient in the food or beverage may be added 0.01 to 20% by weight of the total food or beverage weight, and the health beverage composition may be added in a ratio of 0.02 to 5 g, preferably 0.3 to 1 g, based on 100 ml. have.

The health functional beverage composition of the present invention is not particularly limited in other ingredients other than containing pine wood oil, and may contain various flavoring agents or natural carbohydrates as additional ingredients like conventional beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose; conventional sugars such as disaccharides such as maltose, sucrose, and the like and polysaccharides such as dextrin, cyclodextrin, and the like, and sugar alcohols such as xylitol, sorbitol, erythritol and the like. In addition to the above, natural flavoring agents (taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used as flavoring agents other than those described above. The proportion of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, organic acids, protection It may contain a sexual colloid thickener, a pH adjuster, a stabilizer, a preservative, glycerin, alcohol, a carbonation agent used in carbonated beverages, and the like.

In addition, the composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The proportion of these additives is not critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, antiviral functional cosmetics, multi-purpose cleaners, preservatives, and additives using the composition of the present invention may be prepared and provided.

So far, the present invention has been focused on preferred embodiments.

The embodiments described in this specification and the configurations shown in the drawings relate to one most preferred embodiment of the present invention, and do not represent all of the technical spirit of the present invention, so various equivalents and modifications that can be substituted for them are It should be understood that there may be examples.

Therefore, the present invention is not limited to the presented embodiments, and within the equivalent scope of the technical spirit of the present invention and the technical spirit described in the claims to be described below by those of ordinary skill in the technical field to which the present invention pertains. There may be embodiments in which various modifications and changes are possible.

Claims (6)

It contains pine xylem oil obtained by steam distillation of pine ( Pinus densiflora Siebold & Zucc.) xylem as an active ingredient,
The pine wood oil is,
It is characterized in that it contains 10 ^-3 to 10 ^-2 v/v % concentration for antiviral functionality against influenza A, and 10 ^-2 v/v % concentration for antiviral functionality against Feline coronavirus (FCoV) to, an antiviral composition. The method according to claim 1,
The pine xylem oil inhibits the activity and proliferation of influenza A virus (H1N1), characterized in that it inhibits the activity and proliferation of Feline coronavirus (FCoV), antiviral composition. delete An antiviral food composition comprising the antiviral composition according to claim 1 as an active ingredient. An antiviral cosmetic composition comprising the antiviral composition according to claim 1 as an active ingredient. An antiviral detergent containing the antiviral composition according to claim 1 as an active ingredient.

Images