KR101341532B1 - A composition comprising theanine for treating or preventing bronchitis - Google Patents

Abstract

The present invention relates to a composition comprising theanine for treating or preventing bronchitis. According to the present invention, the composition comprising theanine for treating or preventing bronchitis prevents the activity of immune cells and cytokine.

Description

A composition comprising theanine for treating or preventing bronchitis

The present invention relates to a composition for the prevention or treatment of bronchitis containing thianin.

Bronchitis is an acute and chronic inflammatory disease of the bronchus. Acute bronchitis appears during the course of mechanical or chemical irritation, colds, and various diseases, complaining of chest pain, cough, sputum, etc., but the prognosis is good. Chronic bronchitis is a chronic lung disease in which cough and sputum continue for more than two years. In addition to chronic bronchitis, a sputum cough can last for a long time in a lung disease or airway disease, so to diagnose chronic bronchitis, you should first check for other lung disease or airway disease.

In bronchitis, the inflammatory activity formed in the bronchus is mainly caused by helper T-cells (Th-cells) secreting cytokines. These inflammatory cytokines include TNF-α (tumor necrosis factor-alpha). ), Interferon-gamma (INF-γ), and interleukin such as IL-4, IL-5, and IL-13 (Roh et al., 2008). The cytokines induce replacement of inflammatory cells, eosinophils, inflammation of lung tissue, and the like. On the other hand, as inflammatory cells are activated, expression of related genes is increased by regulating the transcription factors of inflammation-related genes (Barnes and Adcock, 1998), and NF-κB (nuclear factor-κB) is a variety of transcription factors involved in immune and inflammatory responses. (Blackwell and Christman, 1997; Wulczyn et al., 1996). After inflammatory stimulation, I-κB (inhibitor-κB) is phosphorylated to break down, and NF-κB in the cytoplasm enters the nucleus, and cytokines such as IL-1β, IL-6, TNF-α, and cyclooxygenase (COX-2). -2) and induce the expression of a variety of genes, such as enzymes such as matrix metalloproteases (MMPs), fixed substances, acute-phase proteins (Barnes and Karin, 1997). Recently, increased expression of MMP-2 and MMP-9 has been reported due to increased NF-κB activity, including emphysema, acute respiratory distress syndrome, and pulmonary fibrosis. It is associated with airway remodeling in the back, which has also been shown to affect the depletion of collagen type IV in the extracellular matrix (ECM) (Parks and Shapiro). , 2001; Cataldo et al., 2003).

Tianine (theanine) is the highest content of amino acids in tea, giving green tea a sweet and rich flavor. It is used as an ingredient and flavoring agent in diet foods. It can also be used as a flavor enhancer or an additive to improve the taste of tea. Among the 50 kinds of organic compounds in green tea, catechins and amino acids are most closely related to green tea's efficacy and taste. These ingredients vary depending on the type of green tea, cultivation conditions, and harvesting time of tea leaves. Catechins primarily provide a bitter and bitter taste, while amino acids such as thianin provide a rich and sweet taste. Tianine is biosynthesized using glutamine (L-glutamine) and ethylamine at the root of green tea and stored in the leaves of the enzyme, which is chemically decomposed and converted into catechin precursors when exposed to sunlight.

Tianine is also known to have a relaxing effect. In addition, even though coffee contains caffeine like coffee, the reason why it is not excited and stable unlike coffee is because of the tannin. Since green tea is brewed at a lower temperature than coffee, only 60% of caffeine is extracted compared to its actual content, which means that caffeine content is relatively lower than coffee, but green tea contains ingredients that are not found in coffee such as deophyrin, thianin, and catechin. Because these substances combine with caffeine to make insoluble ingredients or inhibit their activity, the side effects of drinking coffee do not appear. In addition, thianine stabilizes and activates the entire nervous system by releasing neurotransmitter chemicals and activating the neurotransmitter system. In addition, thianine is known to have effects such as immune enhancing, lowering blood pressure, lowering blood cholesterol, relieving stress, improving sleep, strengthening concentration, antidepressant treatment, and alcohol detoxification.

On the other hand, the present inventors completed the present invention by confirming the excellent therapeutic effect of bronchitis by inhibiting the secretion of inflammatory cytokines, immune cells, etc., while searching for substances that inhibit bronchitis using a compound derived from natural products Could.

It is not known that thianin has the effect of suppressing bronchial inflammation, and although it has been disclosed that thianin has a therapeutic effect on allergic diseases, it is only confirmed the general allergic reaction in mast cells. Because of this, it can be said to have a different effect from the present invention (Kim et al., 2012). The above-mentioned prior art by Kim et al. Confirms PCA (passive cutaneous anaphylaxis) reaction induced by compound 48/80 (compound 48/80) in mice. In the prior document, DNP-IgE was sensitized to mouse skin. Inducing an IgE-mediated skin response, this experiment only recognizes IgE as an antigen and confirms that the sensitized 'skin allergic response' is reduced by the administration of thianin. In addition, In the prior literature, the expression and secretion of cytokines (TNF-α, IL-6, IL-8 and IL-1β) increased by PMA induction in HMC-1 cells, which are human mast cells, Only general allergic reactions such as antihistamine effects are confirmed, but the inhibitory effect of bronchitis is not confirmed. In contrast, the present invention measures inflammatory cells and inflammatory cytokines secreted from bronchitis caused by bronchial exposure of ovalbumin in bronchoalveolar lavage fluid. In addition, the present invention suggests that TNF-α and IFN-γ (Th1 cytokine), IL-4, IL-5, and IL-13 (Th2 cytokine) are reduced due to the administration of thiin, thereby inhibiting the inflammatory response. In addition, Th2 cytokines stimulate the secretion of IgE and are the main cause of chronic bronchitis, accompanied by airway hypersensitivity and airway remodeling. In addition, in the present invention, by confirming the amount of reduction of inflammatory cells (leukocytes) in the bronchoalveolar lavage fluid after the administration of thiin, it can be said that the present invention is different from the above-mentioned literature, in order to confirm that thianin has an inhibitory effect on bronchial inflammation. have.

On the other hand, although it is disclosed that thianin has an effect on colds (Bukowski and Percival, 2007; Kurihara et al., 2010), the preceding documents do not disclose a direct bronchitis inhibitory effect of thianin. In particular, Bukowski et al., Prior art, discloses that thianin affects the expression of γδ type receptors on T cells. In the absence of thianin, γδ type receptors on T cells are not well expressed. In contrast to the present invention, an immune response (antimicrobial response) did not occur due to an increase in IL-12, IFN-γ, TNF-α, and the like.

Barnes, P. J., Adcock, I. M., 1998. Transcription factors and asthma. Eur. Respir. J., 12, 221 ?? 234. Blackwell and Christman, 1997.The role of nuclear factor-kappa B in cytokine gene regulation. Am. J. Respir. Cell. Mol. Biol., 17, 3 ?? 9. Bukowski and Percival, L-theanine intervention enhances human γδ T lymphocyte function, Nutrition Reviews, 2007, 66 (2), 96 ?? 102. Burrows, 1995. Allergy and the development of asthma and bronchial hyperresponsiveness. Clin. Exp. Allergy, 25, 15 ?? 16. Cataldo et al., 2003. Pathogenic role of matrix metalloproteases and their inhibitors in asthma and chronic obstructive pulmonary disease and therapeutic relevance of matrix metalloproteases inhibitors. Cell. Mol. Biol. (Noisy-le-grand)., 49, 875 ?? 884. Kim et al., Theanine is a candidate amino acid for pharmacological stabilization of mast cells, Amino Acids. 2012. 42. 1609 ?? 1618. Kurihara et al., Effects of L-Cystine and L-Theanine Supplementation on the Common Cold: A Randomized, Double-Blind, and Placebo-Controlled Trial, J. Amino Acid ,. 2010, 307 475, 7. Parks and Shapiro, 2001. Matrix metalloproteinases in lung biology. Respir. Res., 2, 1 ?? 10. Roh et al., 2008. Effects of radix adenophorae and cyclosporine A on an OVA-induced murine model of asthma by suppressing to T cells activity, eosinophilia, and bronchial hyper-responsiveness. Mediat. Inflamm., 781425, 1 ?? 11.

An object of the present invention is to provide a composition for the prevention or treatment of bronchitis containing thianin.

The present invention relates to a composition for the prophylaxis or treatment of bronchitis containing thianin of the formula (1).

[Formula 1]

The bronchitis may be acute bronchitis or chronic bronchitis.

The composition may be a composition having an effect of inhibiting the secretion of inflammatory cytokines or immune cells.

In addition, the present invention relates to a health functional food for the prevention or improvement of bronchitis containing the thiamine of the formula (1). The health functional food may be selected from various foods, health drinks, gum, tea, vitamin complexes and food additives.

Hereinafter, the present invention will be described in detail.

The thianine may be synthesized by conventional methods, and may be extracted from plants such as green tea. The plant extract may be prepared using a mixed solvent of C1 to C4 lower alcohol or water, according to the conventional method, extraction with an organic solvent (alcohol, ether, acetone, etc.), hexane and water distribution, chromatography After the fractionation or purification using a known method used in isolation or extraction of plant components, such as by a single or suitably combined method, such thiamine can be separated.

The chromatography can be carried out using silica gel column chromatography, LH-20 column chromatography, ion exchange resin chromatography, medium pressure liquid chromatography chromatography, thin layer chromatography (TLC), silica gel vacuum liquid chromatography, and high performance liquid chromatography.

On the other hand, the present invention provides a pharmaceutical composition for the prevention or treatment of bronchitis containing thianin. The content of thiamine in the pharmaceutical composition may be appropriately adjusted according to the use form and purpose, the severity of the patient, etc., may be 0.01 to 99.9% by weight, preferably 0.1 to 50% by weight based on the weight of solids, but is not limited thereto. .

In addition, the pharmaceutical composition may be administered in various oral and parenteral dosage forms during actual clinical administration, and when formulated, diluents or excipients such as fillers, weights, binders, wetting agents, disintegrating agents, and surfactants that are commonly used may be used. Can be used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which solid preparations contain at least one excipient in thianine, such as starch, cellulose, calcium carbonate, crospovidone, hard silicic anhydride. , Lactose, magnesium stearate, talc, enzyme treatment stevia, microcrystalline cellulose, magnesium stearate, gelatin and the like can be used in combination. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, and freeze-drying agents. Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like.

The dosage of the pharmaceutical composition may vary depending on the patient's worm, age, sex, health status, diet, administration time, administration method, excretion rate and severity of the disease, at a predetermined time interval according to the judgment of the doctor or pharmacist It may be administered once or several times a day. For example, the daily dose may be 0.001 to 500 mg / kg, preferably 0.1 to 200 mg / kg, based on the active ingredient content.

In another aspect, the present invention provides a dietary supplement for the prevention or improvement of bronchitis containing thianin. The health functional food may be a variety of foods, health drinks, gum, tea, vitamin complex, food additives and the like. The content of thiamine as an active ingredient contained in the health functional food may be appropriately contained depending on the form of the food and the intended use thereof. For example, the thiamine may be added in an amount of 0.01 to 50% by weight of the total food weight. It may be added at a ratio of 0.1 to 10% by weight. In addition, the health beverage composition may be added at a ratio of 0.02 to 10 g, preferably 0.1 to 5 g based on 100 ml. The health beverage composition, in addition to containing the extract as an essential ingredient in the indicated ratio, there is no particular limitation on the liquid component, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like Sugar, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio 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 health functional food of the present invention, various nutritional supplements, vitamins, minerals (electrolytes), Flavoring agents such as synthetic and natural flavoring agents, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin , Alcohols, carbonation agents used in carbonated drinks, etc. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. The components can be used independently or in combination. It is usually such that the ratio of the additives is not so important, but the composition selected in the range of 100 parts by weight of 0.001 to about 20 parts by weight per the present invention.

Tianine, the main component of the composition of the present invention, has been found in animal studies to have no effect in vivo as a result of toxicity tests on standardized herbal compositions.

The present invention relates to a composition for the prevention or treatment of bronchitis containing thianin, the composition is excellent in inhibiting the activity of cytokines and immune cells inducing bronchitis can be usefully used as a therapeutic agent for bronchitis.

1 is a diagram showing the process of inducing a bronchitis animal model of the present invention.
2 is a graph showing that the secretion of TNF-α and INF-γ in bronchoalveolar lavage fluid of bronchiol animal model is suppressed.
3 is a graph showing that the release of IL-4, IL-5 and IL-13 in the bronchial alveolar lavage fluid of the bronchitis animal model is suppressed.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, the intention is to provide an exhaustive, complete, and complete disclosure of the principles of the invention to those skilled in the art.

Example 1. Purchasing Tianine

Tianine was purchased from Tokyo Chemical Industry (TCI) co., Ltd. (Catalog # T0954).

Example 2 Induction of Bronchitis Animal Model

Six-week-old female ICR mice used as experimental animals were purchased one week before the experiment in Daehan Bio (Chungbuk, Korea). The temperature and humidity of the kennel were maintained at an average of 21 ℃, 50%. In addition, the light in the kennel was adjusted to repeat day and night every 12 hours.

To induce a bronchitis model (see FIG. 1), mice were treated with PBS (ckiken egg ovalbumin (50 μg / ml) and albumin (aluminum hydroxide gel, adjuvant, 1 mg / ml) on days 1, 7 and 14. It was dissolved in phosphate buffer saline) and 100 μl each was intraperitoneal injection.

Tianine was dissolved in 100 μl of PBS in 10, 50 and 100 mg / kg / day, and intragasstrically administered daily for 5 days on 12-16 days, and the control group was administered only PBS.

On the last day of Ovalbumin administration, 1 mg / ml Ovalbumin dissolved in PBS was inhaled into the nose (spray using a humidifier) to sensitize the mice (challenge, induce antigen-antibody reactions). Only PBS was allowed to enter. The bronchitis animal model thus made was used for the experiment from the following day.

<Example 3. Measurement of the number of neutrophils, eosinophils and lymphocytes in bronchoalveolar lavage fluid>

Levels of neutrophils, eosinophils and lymphocytes in bronchoalveolar lavage fluid (BALF) act as mediators of bronchial and alveolar inflammation using bronchoalveolar lavage fluid (BALF) in laboratory animals Was measured. To obtain bronchoalveolar lavage fluid, mice were anesthetized, and a cannula was inserted into the trachea while gently rubbing the throat. Immediately after sacrifice, the trachea and lungs were washed five times with 1 ml of PBS to obtain about 0.8 ml of bronchoalveolar lavage. The bronchial alveolar lavage fluid was then centrifuged (400 g, 4 ° C., 5 min) and the supernatant was stored at −70 ° C. for the measurement of cytokines. Cells from bronchoalveolar lavage fluid were washed three times with PBS and resuspended in 100 μl of PBS. The shape of the cells was stained with Giemsa solution and observed under an optical microscope to confirm the status of the differentiated cells. The total number of cells was measured using cytospin and measured in Table 1. The results are shown.

Condition
Control group
Ovalbumin Ovalbumin +
Tianine
10mg / kg Ovalbumin +
Tianine
50 mg / kg Ovalbumin +
Tianine
100 mg / kg
(Unit number of cells:
1 × 10 ⁴ cells / ml) gun
Cell number
14.0 ± 1.00
59.3 ± 6.43
32.7 ± 3.51
27.0 ± 1.00
16.7 ± 2.08  Eosan 5.0 ± 1.00 57.0 ± 5.57 15.0 ± 2.00 10.7 ± 2.08 7.0 ± 1.00 Basophil
5.3 ± 1.53
24.0 ± 4.00
13.0 ± 3.61
9.0 ± 1.00
7.7 ± 0.58  Lymphocyte 12.3 ± 2.08 48.0 ± 2.00 42.0 ± 1.00 36.0 ± 1.00 31.7 ± 1.53 Macrophage 9.7 ± 1.53 29.0 ± 2.00 25.0 ± 1.00 21.3 ± 1.53 17.0 ± 1.00

Referring to Table 1, the total cell number, eosinophils, basophils, lymphocytes, and macrophages were dose-dependently related to thiamine in the group treated with ovarianbumin compared to the group receiving ovalbumin alone. It is confirmed to decrease. Therefore, it can be seen that the inflammatory response in the bronchus is significantly reduced due to the administration of thiamine.

<Example 4. Confirmation of cytokine change amount>

Sandwich enzyme-linked immunosorbent assay (ELISA) was performed to confirm the cytokine changes in the bronchoalveolar lavage fluid of experimental animals. The ELISA kits of BD Biosciences and R & D Systems were purchased and used, and the results are shown in FIGS. 2 and 3. It was.

※ Kit of IL-4 and IL-5: OptEIA Set mouse IL-4, IL-5 (BD Biosciences, San Diego, CA)

※ Kit of TNF-α, INF-γ and IL-13: DuoSet mouse TNF-α, INF-γ, IL-13 (R & D Systems, Minneapolis, MN)

In FIG. 2, it can be seen that thianin inhibits the secretion of TNF-α and INF-γ in bronchial alveolar lavage fluid of a bronchitis animal model in a concentration-dependent manner. In addition, it is confirmed in FIG. 3 that thianin also inhibits the secretion of IL-4, IL-5, and IL-13 in the bronchoalveolar lavage fluid of the bronchitis animal model. Therefore, it can be seen from the results of FIGS. 2 and 3 that the thianin has an excellent inhibitory effect on bronchitis.

Example 5. Toxicity Test

Example 5-1. Acute toxicity

This study was conducted to investigate the toxicity of animals with acute (within 24 hours) ingestion of excessive amounts of thianin and to determine mortality. Twenty ICR mice were injected into each of the control and experimental groups. Only PEG-400 / tween-80 / ethanol (8/1/1, v / v / v) was administered to the control group, and the experimental group was PEG-400 / tween-80 / ethanol at a concentration of 2 g / kg / day for thianine. Dissolved in (8/1/1, v / v / v) and administered orally, respectively. As a result of investigating each mortality rate after 24 hours after administration, it was confirmed that all mice survived in the control group and the experimental group administered with thianine.

Example 5-2. Organ organs toxicity test in experimental group and control group

Long-term toxicity experiments were conducted by administering thianin to C57BL / 6J mice (10 per group) for 8 weeks at each concentration (maximum concentration 2 g / kg / day). Animals of experimental group treated with thianine and control group administered only PEG-400 / tween-80 / ethanol (8/1/1, v / v / v) to investigate the effect on each organ (tissue) of the animal After 8 weeks from the blood was collected and the blood concentrations of glutamate-pyruvate transferase (GPT) and blood urea nitrogen (BUN) were measured using a Select E (Vital Scientific NV, Netherland) instrument. As a result, GPT, which is known to be related to hepatotoxicity, and BUN, which is known to be related to renal toxicity, showed no significant difference compared to the control group. In addition, liver and kidney were cut from each animal, followed by a general tissue section preparation, histological observation with an optical microscope, and no abnormalities were observed in all tissues.

Preparation Example 1. Pharmaceutical Formulations

Formulation Example 1-1. Manufacture of tablets

200 g of tannin was mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. To this mixture was added a 10% gelatin solution, which was pulverized and passed through a 14-mesh sieve. This was dried, and a mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into tablets.

Formulation Example 1-2. Injection preparation

100 g of 1 g of tannin, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water. This solution was placed in a bottle and sterilized by heating at 20 DEG C for 30 minutes.

<Formulation Example 2: Food Preparation>

Formulation Example 2-1. Manufacture of cooking seasonings

Tiannin was added to the cooking seasoning at 1% by weight to prepare a cooking sauce for health promotion.

Formulation Example 2-2. Manufacture of flour food products

Tianine was added to the flour at 0.1% by weight, and the mixture was used to prepare bread, cake, cookies, crackers, and noodles to prepare foods for health promotion.

Preparation Example 2-3. Manufacture of soups and gravies

Tiannin was added to the soup and gravy at 0.1 wt% to prepare soups and gravy.

Formulation Example 2-4. Manufacture of dairy products

Tianine was added to the milk at 0.1% by weight, and the milk was used to make various dairy products such as butter and ice cream.

Formulation Example 2-5. Vegetable juice manufacturing

Tianine was added to 1,000 ml of tomato juice or carrot juice to prepare vegetable juice for health promotion.

Formulation Example 2-6. Manufacture of fruit juice

Tianine was added to 1,000 ml of apple juice or wine to prepare fruit juice for health promotion.

Claims (5)

A composition for the prevention or treatment of acute bronchitis or chronic bronchitis, characterized in that it contains thianine of the formula (1).
[Chemical Formula 1]

delete The method of claim 1,
The composition is a composition for the prevention or treatment of acute bronchitis or chronic bronchitis, characterized in that the effect of inhibiting the secretion of inflammatory cytokines or immune cells. Health functional food for the prevention or improvement of acute bronchitis or chronic bronchitis, characterized in that it contains the thiamine of the formula (1).
[Chemical Formula 1]

5. The method of claim 4,
The health functional food is a health functional food for the prevention or improvement of acute bronchitis or chronic bronchitis, characterized in that selected from various foods, health drinks, gum, tea, vitamin complexes and food additives.

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