KR20190122453A - A pharmaceutical composition for prevention or treatment of respiratory disease comprising Ginsenoside Re - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing and treating respiratory diseases comprising ginsenoside Re. The composition according to the present invention can alleviate symptoms of respiratory diseases caused by rhinovirus infection. Therefore, the composition according to the present invention can be usefully used for preventing and treating respiratory diseases.

Description

A pharmaceutical composition for prevention or treatment of respiratory disease comprising Ginsenoside Re}

The present invention relates to the use of ginsenoside Re for preventing or treating respiratory diseases, and more particularly, to a pharmaceutical composition for preventing or treating respiratory diseases, including ginsenoside Re; A method of treating a respiratory disease comprising administering the pharmaceutical composition; Health functional food; And a composition for inhibiting close splice collapse. Specifically, the present invention relates to the use of Ginsenoside Re for the prevention of respiratory diseases caused by rhinovirus infection.

Respiratory diseases are known to include pathologies that affect organs and tissues involved in gas exchange and respiratory nerves and muscles. Respiratory diseases come in many varieties, the most common of which is the upper respiratory tract infections commonly called colds. Upper respiratory tract infections, which refer to infections of the nasal cavity, pharynx, epiglottis and larynx in the respiratory tract, are caused by viruses and more than 80% of the causes are caused by adenoviruses, influenza viruses, etc. It is known that infection is caused by rhinoviruses.

Rhinovirus (Rhinovirus) is a virus belonging to the family of Phcormaviridae, RNA virus, the form is similar to poliovirus, accompanied by sinusitis, otitis media, cystic fibrosis, bronchitis. As described above, the problem with rhinoviruses is of global interest because it is the largest cause of viral respiratory infections, and research to develop rhinovirus specific antiviral agents is actively underway in the United States.

In Korea, where there is a sharp change in seasonality and a sudden increase in cold patients in each season, there is an urgent need for treatment for upper respiratory tract infections. Therefore, many researchers are studying rhinoviruses that cause upper respiratory tract infections. The mechanism of infection is not clearly identified, so it is not easy to develop a therapeutic agent for rhinovirus infection, and there are drugs prepared by chemical methods or antiviral drugs for many kinds of viruses, but without side effects, rhino No specific treatment for viruses is known.

Meanwhile, ginsenoside, which is a saponin contained in ginseng and a glycoside of ginseng, is a Protopanaxadiol-type or PPD type according to the structure of aglycone. ) Ginsenosides, Protopanaxatriol-type (PPT type) Ginsenosides, and oleanolic acid type (oleanolic acid type) Ginsenosides can be classified into three categories. It is known that the above ginsenosides have been separated.

Among them, ginsenoside Re is a protopanaxatriol-type (PPT type) ginsenoside among dammarane-based saponins, and the immune system strengthening effect ( Experimental parasitology 135.2, 2013: 234-239), cardiovascular Cardiovascular therapeutics (30.4, 2012), antioxidant effects ( European journal of pharmacology 550.1, 2006: 173-179), diabetes and obesity treatment effects ( Biochimica et Biophysica Acta ( BBA ) -Molecular Basis of Disease 1740.3, 2005: 319 -325) has already been reported, but there is no known effect of ginsenoside Re on the inhibition of rhinovirus infection.

Under these backgrounds, the present inventors have conducted extensive research efforts for the treatment of respiratory diseases according to rhinoviruses, and have identified the mechanism of infection of rhinoviruses and confirm that ginsenoside Re inhibits the infection mechanism. The present invention was completed by confirming the effect of improving symptoms related to respiratory disease.

One object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of respiratory diseases containing ginsenoside Re as an active ingredient.

Another object of the present invention is to provide a method for preventing or treating a respiratory disease, comprising administering the pharmaceutical composition to a subject other than a human.

Another object of the present invention to provide a health functional food for the prevention or improvement of respiratory diseases containing ginsenoside Re as an active ingredient.

Another object of the present invention is to provide a composition for inhibiting tight junction breakdown containing ginsenoside Re as an active ingredient.

As one embodiment for solving the above problems, the present invention provides a pharmaceutical composition for the prevention or treatment of respiratory diseases containing ginsenoside Re as an active ingredient.

Hereinafter, a pharmaceutical composition for preventing or treating a respiratory disease containing ginsenoside Re in the present invention will be described in detail.

As used herein, the term “ginsenoside Re” is a protopanaxatriol-type (PPT type) ginsenoside in a dammarane-based saponin, as shown in Formula 1 below, PPT In the basic carbon skeleton of the type, it is a form of ginsenoside in which glucose and rhamnose (Glc-Rha) are bonded to carbon 6 and one glucose is bonded to carbon 20.

[Formula 1]

Ginsenoside means saponin in ginseng. Ginseng saponins have triterpene saponin, a chemical structure that is different from those found in other plants, and because of its unique pharmacological effect, it is called ginsenoside as Ginseng glycoside.

As used herein, the term “respiratory disease” includes a pathological condition that affects organs and tissues involved in gas exchange and respiratory nerves and muscles, but is not limited thereto, and specifically may be an upper respiratory tract infection. More specifically, it may be a cold, pharyngitis, or laryngitis.

The upper respiratory tract infection is a general term for inflammatory diseases caused by infection of the upper respiratory tract mucosa, including the nasal cavity, pharynx, epiglottis and larynx in the respiratory tract, and causes rapid temperature changes, colds, recessive diseases, overwork and weakness. Can be infected through a virus, where a viral infection specifically caused by rhinoviruses may be the main source of infection.

The term "rhinovirus" in the present invention refers to a virus included in the genus Picornaviruses and rhinoviruses, and is currently known as 1 (A, B), from 2 to 113, about 100 antigenic types. Known as the common cold virus, it is susceptible to winter and spring, has an incubation period of 2 to 5 days, and lasts about a week for cold symptoms. Even after healing, the immunity is short and easy to cause re-infection. The rhinoviruses include, but are not limited to, rhinoviruses 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, more specifically rhinovirus 16.

The present inventors have conducted research to treat respiratory diseases caused by rhinovirus infection, confirmed the mechanism of infection of rhinovirus, and confirmed that ginsenoside Re inhibits the infection mechanism, thereby completing the present invention.

In the present invention, after infecting rhinoviruses to non-mucosal epithelial cells to confirm the rhinovirus infection mechanism, the level of tight junction protein and the amount of reactive oxygen species (ROS) production and the NADPH oxidase (NADPH) oxidase (NOX) inhibitors were used to confirm the change.

In the present invention, the term "tight junction" is a kind of intercellular junction that appears mainly in epithelial cells and endothelial cells, and functions as a septum to leak substances between cells constituting a sheet of epithelial cells or endothelial cells. It can be prevented, and acts as a hedge function to partition the cell membrane around the cord-shaped fence in the lipid of the cell membrane and can help maintain the polarity of epithelial or endothelial cells.

In the present invention, the term "NADPH oxidase (NOX)" is a membrane-bound enzyme complex (membrane-bound enzyme complex) is present in the cell membranes of phagocytes to produce superoxide, such as bacteria and fungi It helps to break down microorganisms. Superoxides produced by NOX can form hydrogen peroxide and, moreover, form reactive oxygen species.

As used herein, the term “reactive oxygen species (ROS)” refers to an oxygen species that is more reactive and richer than the normal triplet oxygen ( ³ O ₂ ). Generally, superoxide (O ^2- ), hydrogen peroxide (H ₂ O ₂ ), hydroxy radical (OH) and singlet oxygen ( ¹ O ₂ ) generated from the stepwise reduction of triplet oxygen are defined as active oxygen species. , Alkoxy radicals (RO ·). Highly reactive oxygen compounds, such as peroxy radicals (ROO.) Or ozone (O ₃ ), nitrogen dioxide (NO ₂ ), may also be included in the active oxygen species. Oxygen species are highly oxidative, causing oxidative action on surrounding cell membranes or DNA, which can damage cell structures and further cause cells to lose function or deteriorate.

In a specific embodiment of the present invention, as a result of infecting rhinoviruses to non-mucosal epithelial cells and confirming the level of tight junction protein and ROS production, the rhinovirus infected group has a low level of tight junction protein and an increase in ROS production compared to the normal group. It was confirmed (FIGS. 1 and 2).

In addition, in one specific embodiment of the present invention, as a result of infecting rhinovirus to non-mucosal epithelial cells pretreated with DPI, a NOX inhibitor capable of inhibiting ROS, and confirming the amount of ROS production and the junction protein level, the DPI untreated control was in close contact. While the level of synaptic protein was decreased and ROS production was increased, the NOX inhibitor treatment group was found to have increased protein levels and decreased ROS production (FIGS. 3 and 4).

In a specific embodiment of the present invention, after infecting and cultured rhinoviruses to non-mucosal epithelial cells pretreated with ginsenoside Re and measuring the level of tight junction protein, in the rhinovirus infection control group not treated with ginsenoside Re While the level of tight junction protein was reduced, it was confirmed that the level of tight junction protein was increased in the ginsenoside Re treatment group (FIG. 8).

In one embodiment of the present invention, after inoculating rhinoviruses to non-mucosal epithelial cells pretreated with ginsenoside Re and culturing the amount of ROS produced, the ROS of the rhinovirus infection control group not treated with ginsenoside Re was measured. While the production amount was increased, it was confirmed that the production amount of ROS decreased in the ginsenoside Re treatment group (FIG. 9).

On the other hand, after infection and culture of non-mucosal epithelial cells pretreated with ginsenosides Rc and Rb1, and measuring the level of tight junction protein and ROS production, in the rhinovirus infection control group not treated with ginsenosides Rc and Rb1 Reduced tight junction protein levels and increased ROS production was confirmed to show no difference in the ginsenoside Rc and Rb1 treatment group (FIGS. 11 and 12).

These results indicate that ginsenoside Re has an inhibitory effect on rhinovirus infection, and that all ginsenosides do not exhibit rhinovirus infection inhibition effect.

As used herein, the term "prevention" may mean any action that inhibits or delays the onset of respiratory disease by administering ginsenoside Re according to the present invention to a subject.

As used herein, the term "treatment" may mean any action that improves or benefits the symptoms of respiratory disease by administering the composition of the present invention to a subject suspected of developing a respiratory disease.

The pharmaceutical composition of the present invention may also be used as a single agent, and may be prepared and used as a complex preparation, further including a drug known to have a recognized respiratory disease treatment effect, and formulated using a pharmaceutically acceptable carrier or excipient unit. It may be prepared in a dosage form or incorporated into a multi-dose container.

As used herein, the term "pharmaceutically acceptable carrier" may refer to a carrier or diluent that does not interfere with the biological activity and properties of the compound to be injected without stimulating the organism. The kind of the carrier usable in the present invention is not particularly limited, and any carrier can be used as long as it is a conventionally used and pharmaceutically acceptable carrier in the art. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and the like. These may be used alone or in combination of two or more thereof. The carrier may include a non-naturally occuring carrier.

In addition, if necessary, other conventional additives such as antioxidants, buffers and / or bacteriostatic agents may be added and used, and diluents, dispersants, surfactants, binders, lubricants, and the like may be additionally added to provide a solution such as an aqueous solution, a suspension, an emulsion, or the like. It may be formulated into a use formulation, pills, capsules, granules or tablets.

In addition, the pharmaceutical composition of the present invention may include a pharmaceutically effective amount of ginsenosides Re. As used herein, the term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, generally in an amount of 0.001 to 1000 mg / kg, preferably 0.05 The amount of to 200 mg / kg, more preferably 0.1 to 100 mg / kg may be administered once to several times daily. However, for the purposes of the present invention, the specific therapeutically effective amount for a particular patient is determined by the specific composition, including the type and extent of the reaction to be achieved, whether or not other agents are used in some cases, the age, weight, general health of the patient, It is desirable to apply differently depending on various factors and similar factors well known in the medical field, including sex and diet, time of administration, route of administration and rate of composition, duration of treatment, drugs used with or co-specific with the specific composition.

The pharmaceutical compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. And single or multiple administrations. In consideration of all the above factors, it is important to administer an amount that can achieve the maximum effect in a minimum amount without causing side effects, and can be easily determined by those skilled in the art.

The term "administration" in the present invention means introducing the pharmaceutical composition of the present invention to a patient in any suitable manner, and the route of administration of the composition of the present invention may be varied orally or parenterally as long as the target tissue can be reached. It can be administered via the route.

The mode of administration of the pharmaceutical composition according to the present invention is not particularly limited, and may be in accordance with methods commonly used in the art. As a non-limiting example of the mode of administration, the composition may be administered by oral or parenteral administration. The pharmaceutical compositions according to the invention may be prepared in various formulations depending on the desired mode of administration.

The frequency of administration of the composition of the present invention is not particularly limited, but may be administered once a day or several times in divided doses.

As used herein, the term 'pharmaceutically acceptable salt' refers to a formulation that does not impair the biological activity and physical properties of the ginsenoside Re administered. The pharmaceutically acceptable salts are acids that form non-toxic acid addition salts containing pharmaceutically acceptable anions, such as inorganic acids, such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, and tartaric acid. Organic carbonic acid, such as formic acid, citric acid, acetic acid, trichloroacetic acid, trichloroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicylic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfate Acid addition salts formed by sulfonic acids and the like such as phonic acid and the like. For example, pharmaceutically acceptable carboxylic acid salts include metal salts or alkaline earth metal salts formed by lithium, sodium, potassium, calcium, magnesium, amino acid salts such as lysine, arginine, guanidine, dicyclohexylamine, N Organic salts such as -methyl-D-glucamine, tris (hydroxymethyl) methylamine, diethanolamine, choline and triethylamine and the like.

In another aspect, the present invention provides a method for preventing or treating respiratory disease, comprising administering ginsenoside Re to a subject other than a human.

The terms "ginsenoside Re" and "respiratory disease" in the present invention are as described above.

As used herein, the term "individual" may mean any animal, including a human, who has or is likely to develop a respiratory disease. The animal may be a mammal such as, but not limited to, a human, a cow, a horse, a sheep, a pig, a goat, a camel, a antelope, a dog, a cat, and the like, which require treatment of similar symptoms.

The prophylactic or therapeutic method of the present invention may specifically include administering the composition in a pharmaceutically effective amount to a subject having or at risk of developing a respiratory disease.

The term "administration" in the present invention means introducing the pharmaceutical composition of the present invention to a patient in any suitable manner, and the route of administration of the composition of the present invention may be varied orally or parenterally as long as the target tissue can be reached. It can be administered via the route.

As another aspect, the present invention provides a health functional food for the prevention or improvement of respiratory diseases containing ginsenoside Re as an active ingredient.

The terms "ginsenoside Re" and "respiratory disease" in the present invention are as described above.

Since ginsenoside Re has been used for a long time as a natural substance and has been proved to be stable, it can be prepared and consumed in the form of a food that can prevent or improve respiratory diseases while having common sense.

Functional food is the same term as food for special health use (FOSHU), and means foods that have high medical effects and medical effects that are processed so that their bioregulatory functions are efficiently displayed in addition to nutrition. In addition, the food may be prepared in various forms such as tablets, capsules, powders, granules, liquids, pills, etc. in order to obtain useful effects in preventing or improving respiratory diseases.

The food composition of the present invention may further comprise a food acceptable carrier.

There is no restriction | limiting in particular in the kind of food which can add the composition containing ginsenoside Re of this invention, For example, there are various drinks, a gum, tea, a vitamin complex, a dietary supplement, etc. The food composition may be added to other ingredients that do not interfere with the effect of preventing or improving respiratory diseases, the kind is not particularly limited. For example, various herbal extracts, food acceptable additives, natural carbohydrates, and the like may be contained as additional ingredients, such as conventional foods.

The food supplement additive is added to prepare the health functional food of each formulation can be used by those skilled in the art as appropriate. For example, various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic and natural flavors, colorants and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters , Stabilizers, preservatives, glycerin, alcohols, carbonation agents used in the carbonated beverage, and the like, but the type is not limited by the above examples.

At this time, the content of the extract included in the food is not particularly limited, but may be included in 0.01 to 100% by weight, more preferably 1 to 80% by weight relative to the total weight of the food composition.

When the food is a beverage, it may be included in a ratio of 1 to 30 g, preferably 3 to 20 g based on 100 ml. In addition, the composition may include additional ingredients that are commonly used in food compositions to improve the smell, taste, time and the like. For example, it may include vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, and the like. In addition, it may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu). It may also contain amino acids such as lysine, tryptophan, cysteine, valine and the like. In addition, preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), fungicides (bleaching powder and highly bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisol (BHA), butylhydroxytoluene ( BHT), etc.), colorants (such as tar pigments), colorants (such as sodium nitrite, sodium nitrite), bleach (sodium sulfite), seasonings (such as MSG glutamate), sweeteners (ducin, cyclate, saccharin, sodium, etc.) Food additives such as flavors (vanillin, lactones, etc.), swelling agents (alum, potassium D-tartrate, etc.), reinforcing agents, emulsifiers, thickeners (pigments), coatings, gum herbicides, foam inhibitors, solvents, improvers, etc. ) Can be added. The additive is selected according to the type of food and used in an appropriate amount.

The health functional food of the present invention can be prepared by a method commonly used in the art, and the preparation can be prepared by adding raw materials and ingredients commonly added in the art. In addition, unlike the general medicine has the advantage that there is no side effect that can occur when taking a long-term use of the drug with food as a raw material, can be excellent in portability.

As another aspect, the present invention provides a composition for inhibiting close splice collapse, comprising ginsenoside Re as an active ingredient.

The terms "ginsenoside Re" and "close contact" in the present invention are as described above.

As used herein, the term “inhibition of disintegration” may mean inhibiting degradation by maintaining binding of a protein.

In one embodiment of the present invention, the rhinoviruses were infected with non-mucosal epithelial cells pretreated with ginsenoside Re and the level of tight junction protein was measured. As a result, the tight junction protein was confirmed to be increased. Therefore, this invention can suggest that ginsenoside Re can be used as a composition for suppressing close splice collapse.

The composition according to the invention can ameliorate the symptoms of respiratory disease caused by rhinovirus infection. Therefore, the composition according to the present invention can be usefully used for the prevention and treatment of respiratory diseases.

1 is a graph of tight junction-related protein measurements to confirm tight junction collapse after rhinovirus infection in non-mucosal epithelial cells; A. Occludion, B. ZO-1, C. Claudin, D. E-cadherin; RV 24-72h: 24-72 hours of culture after rhinovirus infection.
Figure 2 is a graph confirming the amount of ROS generated after rhinovirus infection to non-mucosal epithelial cells; MEDIUM: normal group, RV-16: rhinovirus infected group.
Figure 3 is a graph measuring ROS production following rhinovirus infection after NOX activity inhibitor (DPI) treatment; A. ROS production after DPI treatment, B. ROS production after rotenone treatment, C. ROS production using microscope after DPI treatment, MEDIUM: normal group, RV-16: rhinovirus infection group, DPI 25-50μM: DPI 25 ~ 50 µM treatment group, Rotenone 15-30 µM: Rotenone 15-30 µM treatment group.
Figure 4 is a graph measuring tight junction protein levels following rhinovirus infection after DPI treatment; A. ZO-1, B. E-cadherin, C. Occludin. D. Claudin; MEDIUM: normal group, DPI 25-50 μM: DPI 25-50 μM treated group, RV 72h: 72 hours incubation after rhinovirus infection.
5 is a graph confirming the phosphatase activity and phosphorylated tyrosine protein level according to rhinovirus infection; A. phosphatase activity graph, B. phosphorylated tyrosine protein measurement graph; Medium: normal group, RV16 (48h): cultured 48 hours after rhinovirus infection, RV 24-72h: cultured 24-72 hours after rhinovirus infection, p-tyrosine: phosphorylated tyrosine.
6 is a graph showing phosphatase activity and phosphorylated tyrosine protein levels following rhinovirus infection after DPI pretreatment; A. phosphatase activity graph, B. phosphorylated tyrosine protein measurement graph; Medium: normal group, DPI 25-50 μM: DPI 25-50 μM treatment group.
7 is a graph showing phosphatase activity and tight junction protein levels following rhinovirus infection after phosphatase inhibitor (PAO) pretreatment; A. phosphatase activity graph, B. tightly coupled protein measurement graph; PAO 1-500 nM: PAO 1-500 nM treatment group.
Figure 8 is a graph showing the tight junction protein level following rhinovirus infection after ginsenoside Re pretreatment; Re (50-100 μg / ml): Ginsenoside Re 50-100 μg / ml treated group.
9 is a graph showing the amount of ROS produced by rhinovirus infection after ginsenoside Re pretreatment.
10 is a graph showing phosphatase activity and phosphorylated tyrosine protein levels following rhinovirus infection after ginsenoside Re pretreatment.
FIG. 11 is a graph showing tight junction protein levels following rhinovirus infection after pretreatment of ginsenosides Rc and Rb1, respectively; Rc (50-100 μg / ml): Ginsenoside Rc 50-100 μg / ml treated group, Rb1 (50-100 μg / ml): Ginsenoside Rb1 50-100 μg / ml treated group.
12 is a graph showing the amount of ROS produced by rhinovirus infection after pretreatment of ginsenosides Rc and Rb1, respectively.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example  One. Nasal mucosa  Preparation of Epithelial Cells and Rhinoviruses

Non-mucosal epithelial cells and rhinoviruses were prepared to identify changes in rhinovirus infection in non-mucosal epithelial cells. In the case of nasal mucosal epithelial cells, the nasal epithelial cells were obtained from chronic hypertrophic rhinitis patients who underwent inferior turbinate surgery, and then treated with 1% pronase using 1% pronase. Separated. For rhinoviruses, HeLa cells were infected with rhinovirus 16 (RV-16) and left at 33 ° C. for 7-10 days until cell lesion effect (CPE) was formed and concentrated to increase virus titer. . The amount of sample required to infect 50% HeLa cells after concentration (50% tissue culture infection dose, TCID ₅₀ ) was measured and the virus titer was expressed as TCID ₅₀ / ml using Karber's formula.

Experimental Example  1. Confirm changes due to rhinovirus infection

1-1. Confirmation of tight junction protein change by rhinovirus infection

In order to confirm the change in the tight junction protein caused by rhinovirus infection, Western blot was performed by extracting the tight junction protein of non-mucosal epithelial cells cultured for 24 to 72 hours at 37 ° C. and 5% CO _2. It was.

As a result, as shown in Figure 1, it was confirmed that the tight junction protein decreases with time after infection with rhinovirus. From this, it was confirmed that the rhinovirus dissociated the tight junction protein to cause the tight junction collapse.

1-2. Caused by rhinovirus infection Reactive oxygen species ( ROS Output measurement

In order to confirm the increase in ROS production caused by rhinovirus infection, non-mucosal epithelial cells cultured in BEGM medium were infected with rhinovirus and incubated for 24 hours at 37 ° C. and 5% CO ₂ , followed by flow cytometry and DCFDA dye. ROS production was confirmed through a microscope.

As a result, as shown in Figure 2, it was confirmed that the intracellular ROS increased after 24 hours of culture, rhinovirus infection increases the production of ROS.

Experimental Example  2. rhinovirus infection NADPH  Oxidase ( NADPH oxidase , NOX ) Impact on

2-1. After DPI processing ROS  Measure changes in production

After pretreatment of the NOX inhibitor, to determine whether ROS production by rhinovirus infection can be inhibited, the NOX activity inhibitor (DPI) and the mitochondrial electron transfer inhibitor rotenone (rotenone), respectively, were pretreated into the nasal mucosal epithelial cells, followed by rhino After infecting the virus and incubating at 37 ° C., 5% CO ₂ for 24 hours, the difference in ROS production was determined.

As a result, as shown in Fig. 3, when the inhibitor was used, the rotenone treated group was not different from the virus infected group, but the DPI treated group confirmed that the ROS increased by the rhinovirus was decreased, indicating that NOX activity was increased by ROS. It was confirmed that it is associated with the production.

2-2. NOX  Measurement of tight junction protein changes after inhibitor treatment

After pretreatment of the inhibitor of NOX, to determine whether it is possible to suppress the decrease in the tight junction protein caused by rhinoviruses, 37 ° C., 5% CO after infecting RV-16 with non-mucosal epithelial cells pretreated with DPI for each concentration. After culturing for 72 hours at ₂ conditions, the contact protein was extracted and Western blot was performed.

As a result, as shown in Figure 4, it was confirmed that the tight junction protein decreased during rhinovirus infection increased after DPI treatment, it was confirmed that the breakdown of the tight junction is associated with the activity of NOX.

Experimental Example  3. Rhinovirus Infection of Phosphorylase and Phosphorylation Tyrosine  Identify the impact on protein

3-1. Phosphorylase Activity and Phosphorylation by Rhinovirus Infection Tyrosine  Protein level measurement

In order to determine the activity of phosphatase and protein level of phosphorylated tyrosine by rhinovirus infection, non-mucosal epithelial cells cultured in BEGM medium were infected with rhinovirus for 24 to 72 hours at 37 ° C., 5% CO ₂ conditions. After incubation, the activity of phosphatase was measured using a phosphatase activity kit, and the phosphorylated tyrosine protein level was measured using a Western blot.

As a result, as shown in FIG. 5, the activity of phosphatase was decreased during rhinovirus infection, whereas the phosphorylated tyrosine protein was increased.

3-2. NOX  Phosphorylase Activity and Phosphorylation after Inhibitor Treatment Tyrosine  Protein level measurement

After pretreatment of NOX inhibitors, 37 ° C after infection of rhinovirus epithelial cells pre-treated with DPI at different concentrations to determine whether phosphatase activity and phosphorylated tyrosine protein levels changed by rhinovirus infection were affected. After culturing for 72 hours at 5% CO ₂ condition, the activity of phosphatase was measured using a phosphatase activity kit, and the phosphorylated tyrosine protein level was measured using a Western blot.

As a result, as shown in Figure 6, the NOX inhibitor treatment group was confirmed that the phosphatase activity decreased by rhinovirus infection was increased, and that the increased phosphorylated tyrosine protein was decreased, the phosphatase according to rhinovirus infection And it was confirmed that the effect on phosphorylated tyrosine is associated with NOX activity.

3-3. Measurement of phosphatase activity and changes in tight junction protein after phosphatase inhibitor treatment

To determine whether phosphatase affects rhinovirus infection and tight junctions, pretreatment with phosphatase inhibitors at each concentration in non-mucosal epithelial cells cultured in BEGM medium, followed by infection with rhinovirus, After 48 hours of incubation at 5% CO ₂ , the activity of phosphatase was measured and the level of tight junction protein was measured.

As a result, as shown in Figure 7, the activity of the phosphatase was decreased, confirming that the level of tight junction protein also decreased, confirming that the activity of phosphatase is also associated with the tight junction collapse due to rhinovirus infection. It was.

Incorporating the contents of Experimental Examples 1 to 3, rhinovirus infection induces NOX activity, which increases the amount of ROS produced, decreases the activity of phosphatase, increases phosphorylated tyrosine protein, and finally breaks the tight junction. I could infer what caused it.

Experimental Example  4. Confirmation of ginsenoside Re according to the inhibitory effect of rhinovirus infection

4-1. Changes in tight junction protein after ginsenoside Re treatment

After pre-treatment with ginsenoside Re, to determine whether it is possible to suppress the decrease in the tight junction protein caused by rhinovirus, 37 ℃, After culturing for 72 hours at 5% CO ₂ conditions, the contact protein was extracted and Western blot was performed.

As a result, as shown in Figure 8, the ginsenoside Re treatment group confirmed that the increase in the tight junction protein reduced during rhinovirus infection confirmed that the ginsenoside Re inhibits the tight junction collapse by the rhinovirus.

4-2. After Ginsenoside Re Treatment ROS  Confirmation of change in generation

After pretreatment of ginsenoside Re, to determine whether ROS production by rhinovirus infection can be inhibited, pretreatment of ginsenoside Re to non-mucosal epithelial cells, followed by infection with rhinovirus at 37 ° C., 5% CO ₂ conditions After culturing for 24 hours at ROS production was confirmed.

As a result, as shown in Figure 9, it was confirmed that the ROS increased by rhinovirus infection decreased in the ginsenoside Re treatment group, confirming that ginsenoside Re inhibits the increase in ROS production by rhinoviruses.

4-3. Phosphorylase Activity and Phosphorylation after Ginsenoside Re Treatment Tyrosine  Identify protein changes

After pretreatment with ginsenoside Re, non-mucosal epithelial cells pretreated with ginsenoside Re for each concentration were examined for rhinoviruses to determine whether the phosphatase activity and phosphorylated tyrosine protein levels changed by rhinovirus infection. After infection, the cells were incubated for 48 to 72 hours at 37 ° C. and 5% CO ₂ , and the phosphatase activity was measured using a phosphatase activity kit, and the phosphorylated tyrosine protein level was measured using a Western blot. It was.

As a result, as shown in Figure 10, the ginsenoside Re treatment group was found to increase the phosphatase activity decreased by rhinovirus infection, increased phosphorylated tyrosine protein was reduced to suppress the change by rhinovirus It confirmed that it did.

According to the contents of Experimental Example 4, the treatment of ginsenoside Re improves ROS production, phosphatase activity, phosphorylated tyrosine protein, and tightly-coupled protein, which were shown as symptoms of rhinovirus infection. It was confirmed that ginsenoside Re has an inhibitory effect on rhinovirus infection symptoms. This suggests that ginsenoside Re has a prophylactic or therapeutic effect on respiratory diseases caused by rhinovirus infection.

Comparative example  1. Confirm the effect of inhibiting the symptoms of rhinovirus infection of other ginsenosides

1-1. Ginsenoside Rc  And Rb1  Confirmation of tight junction protein changes after treatment

After pretreatment with ginsenosides Rc and Rb1, in order to confirm the change in the tight junction protein due to rhinovirus infection, non-mucosal epithelial cells pretreated with each ginsenoside Rc and Rb1 were infected with rhinovirus at 37 ° C., After culturing for 72 hours at 5% CO ₂ conditions, the contact protein was extracted and Western blot was performed.

As a result, as shown in Figure 11, the treatment group of ginsenosides Rc and Rb1 confirmed that there is no change in the tight junction protein that was reduced by rhinovirus infection.

1-2. Ginsenoside Rc  And Rb1  After treatment ROS  Confirmation of change in generation

After pretreatment of ginsenosides Rc and Rb1, to determine whether ROS production by rhinovirus infection can be inhibited, pretreatment of ginsenoside Re into non-mucosal epithelial cells followed by infection of rhinoviruses at 37 ° C., 5% CO After culturing for 24 hours under ₂ conditions, ROS production was confirmed.

As a result, as shown in Figure 12, the treatment group of ginsenosides Rc and Rb1 confirmed that there was no change in the amount of ROS production increased by rhinovirus infection.

Taken together, ginsenoside Re has the effect of suppressing rhinovirus infection symptoms and has a prophylactic or therapeutic effect against respiratory diseases caused by rhinoviruses such as colds, pharyngitis and laryngitis, whereas ginsenosides Rc and Rb1 have an effect. Ginsenoside Re was specifically found to be usable as a pharmaceutical composition for the prevention or treatment of respiratory diseases caused by rhinoviruses.

From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, the embodiments described above are to be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

Claims (8)

Pharmaceutical composition for the prevention or treatment of viral respiratory diseases caused by rhinoviruses comprising ginsenoside Re as an active ingredient.
The method of claim 1,
The respiratory disease is an upper respiratory tract infection, including colds, pharyngitis, laryngitis.
The method of claim 1,
The ginsenoside Re is to inhibit the activity of NOX increased by rhinoviruses and inhibit the breakdown of the close junction, pharmaceutical composition.
A method for preventing or treating a viral respiratory disease caused by rhinoviruses, comprising administering the pharmaceutical composition of claim 1 to an individual other than a human.
The method of claim 4, wherein
The respiratory disease is an upper respiratory tract infection including a cold, pharyngitis, laryngitis, method of preventing or treating respiratory disease.
Health functional food for the prevention or improvement of viral respiratory disease caused by rhinovirus containing ginsenoside Re as an active ingredient.
The method of claim 6,
The respiratory disease is an upper respiratory tract infection including a cold, pharyngitis, laryngitis, health functional foods for the prevention or improvement of respiratory diseases.
A tight junction decay suppression composition comprising ginsenoside Re as an active ingredient.

Images