KR102535396B1 - Composition for preventing or treating respiratory function damage caused by fine dust using citrus fruits peel and method for preparing the same - Google Patents

Abstract

The present invention relates to a composition for preventing and improving fine dust-stimulated respiratory diseases using an extract of the peel of a tangerine genus plant as an active ingredient and a method for preparing the same.

Description

Method for preparing a composition for preventing or treating respiratory function damage caused by fine dust using citrus fruits peel and method for preparing the same}

The present invention relates to a composition for preventing and improving fine dust-stimulated respiratory diseases using an extract of the peel of a tangerine genus plant as an active ingredient, and a method for manufacturing the same, and more particularly, to prevention of fine dust-stimulated respiratory diseases obtained by hot water extraction of citron peel or citron peel. And it relates to a composition for improvement and a method for preparing the same.

Since the 1960s, Korea has been rapidly industrialized and urbanized due to the promotion of economic development, the development and distribution of science and technology, and industrialization. It is a serious situation. As data supporting this, a report was published stating that the level of air pollution in Korea is the worst among OECD countries and the 26th most serious in the world (2019 World Air Quality Report, IQ Air (Switzerland)).

The report found that 21 out of 30 air polluting cities are located in India, and all the rest are in Asia. Similar to Korea, China, which is adjacent to Korea, has rapid industrialization, industrialization, and dependence on coal at over 70%, and yellow dust and smog generated in dry areas in Mongolia are combined to harm us. As a result, fine dust advisories and warnings are increasing in Korea.

Fine dust is caused by combustion of coal fuel or has various causes such as automobile exhaust gas and road dust, but mainly has very complex components such as sulfur oxides, lead, ozone, carbon monoxide, and nitrogen oxides, and floats in the air.

The composition and size of fine dust are very diverse, but it can be divided into ultra-fine dust with a diameter of less than 2.5 μm called PM2.5 and fine dust with a diameter of less than 10 μM called PM10. It has been reported to have adverse effects on organs and parts.

In particular, the effect on the respiratory system that comes into contact with fine dust for the first time is mainly caused by causing an inflammatory reaction in the bronchioles. This stimulation is well known as a cause of chronic bronchitis, asthma, and chronic obstructive respiratory disease.

In addition, lung damage and pulmonary edema are well known as lung diseases caused by fine dust, and it has been found that these diseases can be caused by oxidative stress caused by fine dust.

Moreover, it has been reported that fine dust interferes with the self-defense mechanism of lung cells, reducing or losing the ability to cope with various bacteria that invade lung cells, which causes various respiratory infections.

It has been reported that diseases caused by exposure to fine dust are not limited to the respiratory system, but are related to cardiovascular diseases, cancer occurrence, and an increase in mortality. 2015 Fact Sheet on Pollution and Cardiovascular Disease (American Heart Association)

As such, it was predicted that due to the threat to public health caused by air pollution, especially fine dust, the social cost loss would reach 10 trillion won, reaching 20 trillion won by 2060 (Bae Jeong-hwan, Professor of Economics, Chonnam National University, 2017). Efforts and investments are continuously being made to improve the environment worldwide, and it is also necessary to develop food and medicines that prevent or improve various respiratory diseases caused by fine dust.

Therefore, there is a need to prevent and improve diseases caused by fine dust by providing a composition with fewer side effects and excellent functions by using beneficial components of natural products.

Korean Patent Publication No. 10-2019-0127583 Korean Registered Patent No. 10-1705547

Yang W, Omaye ST (2009) Air pollutants, oxidative stress and human health. Mutat Res 674:45-54

The present invention has been derived from the above needs, and the present invention is to provide a composition for improving fine dust-stimulated respiratory diseases using an extract of the peel of a tangerine genus plant as an active ingredient and a method for preparing the same.

In order to solve the above problems, the present invention provides a solution to the following problems.

One aspect of the present invention provides a pharmaceutical composition for preventing or treating fine dust-stimulated respiratory diseases, comprising an extract of a plant of the genus Citrus as an active ingredient.

In one aspect of the present invention, the extract provides a pharmaceutical composition for preventing or treating fine dust-stimulated respiratory diseases, which is obtained by extracting water, alcohol having 1 to 5 carbon atoms, or an aqueous solution of alcohol having 1 to 5 carbon atoms as an extraction solvent. .

In one aspect of the present invention, the extract provides a water extract, a pharmaceutical composition for preventing or treating fine dust-stimulated respiratory diseases.

In one aspect of the present invention, the extract is extracted for 20 minutes to 10 hours at 50 ° C. to 150 ° C., providing a pharmaceutical composition for preventing or treating fine dust-stimulated respiratory diseases.

In one aspect of the present invention, the plants of the genus Citrus are pomelo (Citrus maxima), citron (Citrus medica), mandarin (Citrus reticulate), tangerine (Citrus trifoliate), golden persimmon (Citrus japonica), grapefruit (Citrus paradisi), citron (Citrus junos ) , lemon (Citrus limon), lime (Citrus aurantifolia), tangerine (Citrus unshiu), satsuma mandarin, and sweet orange (Citrus sinensis), which is any one or more of fine dust-stimulated respiratory disease prevention or treatment It provides a pharmaceutical composition for use.

In one aspect of the present invention, the plant of the genus Citrus is a fruit of the plant, providing a pharmaceutical composition for preventing or treating fine dust-stimulated respiratory diseases.

Another aspect of the present invention provides a food composition for preventing or improving fine dust-stimulated respiratory diseases, comprising an extract of a plant of the genus Citrus as an active ingredient.

In another aspect of the present invention, the extract provides a food composition for preventing or improving fine dust-stimulated respiratory diseases, wherein the extract is obtained by extracting water, alcohol having 1 to 5 carbon atoms, or an aqueous solution of alcohol having 1 to 5 carbon atoms as an extraction solvent. .

In another aspect of the present invention, the extract provides a water extract, a food composition for preventing or improving fine dust-stimulated respiratory diseases.

In another aspect of the present invention, the extract is extracted for 20 minutes to 10 hours at 50 ° C. to 150 ° C., providing a food composition for preventing or improving fine dust-stimulating respiratory diseases.

In another aspect of the present invention, the plants of the genus Citrus are pomelo (Citrus maxima), citron (Citrus medica), mandarin (Citrus reticulate), tangerine (Citrus trifoliate), golden persimmon (Citrus japonica), grapefruit (Citrus paradisi), citron (Citrus junos ) , Lemon (Citrus limon), Lime (Citrus aurantifolia), Tangerine (Citrus unshiu), Wenzhou tangerine (satsuma mandarin), and Sweet orange (Citrus sinensis), any one or more, to prevent or improve fine dust-stimulated respiratory diseases It provides a food composition for use.

In another aspect of the present invention, the plant of the genus Citrus is a fruit of the plant, providing a food composition for preventing or improving fine dust-stimulated respiratory diseases.

Another aspect of the present invention provides a method for preparing the pharmaceutical composition or food composition.

The present invention relates to a composition for preventing and improving fine dust-stimulated respiratory diseases containing tangerine rind, preferably citron rind or citron peel, as an active ingredient, and a method for manufacturing the same. It inhibits the expression of inflammatory factors TNF-α, IL-6, and IL-1β, and the expression of c-Caspsae, which is a signal transduction mechanism for cell death or apoptosis of lung cells, and activates tissue and intracellular reactive oxygen species (ROS). ) can be effectively used to prevent and improve respiratory diseases stimulated by fine dust. In addition, a specific manufacturing method is presented in order to more efficiently complete the present invention for preventing and improving the respiratory disease.

1 is a result of confirming the antioxidant effect of the rind extract of a plant of the genus Tangerine. 1(A) is ABTS, and (B) is the DPPH result.
Figure 2 is the result of confirming the cell protective effect of the rind extract of the tangerine genus plant against the death of lung cells induced by fine dust.
Figure 3 is the result of confirming the effect of inhibiting the generation of reactive oxygen species (ROS) due to intracellular oxidative stress of the rind extract of Tangerine genus.
Figure 4 is the result of confirming the preventive effect of citron peel and citron peel extract on lung damage caused by fine dust (PM10) in experimental animals.
Figure 5 is the result of confirming the effect of inhibiting the expression of inflammatory cytokines (TNF-α, IL-1β) by citron peel and citron peel extract in bronchial lavage fluid (BALF) of experimental animals.
Figure 6 is the result of confirming the effect of inhibiting the expression of apoptosis factors (c-Caspase-9, c-Caspase-3) by citron peel and citron gourd extract in lung tissues of experimental animals.
Figure 7 is the result of confirming the inhibitory effect of inflammatory cytokine (IL-6) expression by citron peel and citron gourd extract in the lung tissue of experimental animals.

Unless otherwise specified, all numbers, values and/or expressions expressing components, reaction conditions, or amounts of components used herein are intended to indicate that such numbers, among other things, are inherently different from the measurement taken to obtain such values. Since these are approximations that reflect uncertainty, they should be understood as being qualified by the term "about" in all cases. Also, when numerical ranges are disclosed herein, such ranges are contiguous and include all values from the minimum value of such range to the maximum value inclusive, unless otherwise indicated. Furthermore, where such ranges refer to integers, all integers from the minimum value to the maximum value inclusive are included unless otherwise indicated.

In this specification, where ranges are stated for a variable, it will be understood that the variable includes all values within the stated range inclusive of the stated endpoints of the range. For example, a range of "5 to 10" includes values of 5, 6, 7, 8, 9, and 10, as well as any subrange of 6 to 10, 7 to 10, 6 to 9, 7 to 9, and the like. inclusive, as well as any value between integers that fall within the scope of the stated range, such as 5.5, 6.5, 7.5, 5.5 to 8.5 and 6.5 to 9, and the like. Also, for example, the range of "10% to 30%" includes values such as 10%, 11%, 12%, 13%, etc., and all integers up to and including 30%, as well as values from 10% to 15%, 12% to 12%, etc. It will be understood to include any sub-range, such as 18%, 20% to 30%, and the like, as well as any value between reasonable integers within the scope of the stated range, such as 10.5%, 15.5%, 25.5%, and the like.

Hereinafter, various aspects of the present invention will be described in detail.

One aspect of the present invention provides a pharmaceutical composition for preventing or treating fine dust-stimulated respiratory diseases, comprising an extract of a plant of the genus Citrus as an active ingredient.

In one aspect of the present invention, the extract provides a pharmaceutical composition for preventing or treating fine dust-stimulated respiratory diseases, which is obtained by extracting water, alcohol having 1 to 5 carbon atoms, or an aqueous solution of alcohol having 1 to 5 carbon atoms as an extraction solvent. .

In one aspect of the present invention, the extract provides a water extract, a pharmaceutical composition for preventing or treating fine dust-stimulated respiratory diseases.

In one aspect of the present invention, the extract is extracted for 20 minutes to 10 hours at 50 ° C. to 150 ° C., providing a pharmaceutical composition for preventing or treating fine dust-stimulated respiratory diseases.

In one aspect of the present invention, the plants of the genus Citrus are pomelo (Citrus maxima), citron (Citrus medica), mandarin (Citrus reticulate), tangerine (Citrus trifoliate), golden persimmon (Citrus japonica), grapefruit (Citrus paradisi), citron (Citrus junos ) , lemon (Citrus limon), lime (Citrus aurantifolia), tangerine (Citrus unshiu), satsuma mandarin, and sweet orange (Citrus sinensis), which is any one or more of fine dust-stimulated respiratory disease prevention or treatment It provides a pharmaceutical composition for use.

In one aspect of the present invention, the plant of the genus Citrus is a fruit of the plant, providing a pharmaceutical composition for preventing or treating fine dust-stimulated respiratory diseases.

The pharmaceutical composition according to one aspect of the present invention may be in various oral or parenteral dosage forms. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, soft or hard capsules, etc. These solid preparations contain at least one excipient such as starch, calcium carbonate and sucrose in one or more compounds. Alternatively, it is prepared by mixing lactose and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The pharmaceutical composition according to one aspect of the present invention may be administered parenterally or orally as desired, and may be administered in an amount of 0.1 to 500 mg, specifically 1 to 100 mg per 1 kg of body weight per day, once to several times to be administered. Can be administered in divided doses. The dosage for a specific patient may vary depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, severity of the disease, and the like.

The pharmaceutical composition according to one aspect of the present invention, according to a conventional method, oral formulations such as powders, granules, tablets, soft or hard capsules, suspensions, emulsions, syrups, drinks, aerosols, ointments, creams, etc. It can be formulated and used in any form suitable for pharmaceutical preparations, including external preparations, suppositories, injections and sterile injection solutions, etc., and can be specifically formulated and used in the form of injections or external preparations for the skin.

The pharmaceutical composition according to one aspect of the present invention can be administered to mammals such as rats, mice, livestock, and humans through various routes such as parenteral and oral, and all methods of administration can be expected, for example , orally, transdermally, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The pharmaceutical composition according to one aspect of the present invention may be administered through various routes that can be easily applied by those skilled in the art. In particular, the pharmaceutical composition according to the present specification may be administered as a skin external agent through a route applied to the skin surface.

Another aspect of the present invention provides a food composition for preventing or improving fine dust-stimulated respiratory diseases, comprising an extract of a plant of the genus Citrus as an active ingredient.

In another aspect of the present invention, the extract provides a food composition for preventing or improving fine dust-stimulated respiratory diseases, wherein the extract is obtained by extracting water, alcohol having 1 to 5 carbon atoms, or an aqueous solution of alcohol having 1 to 5 carbon atoms as an extraction solvent. .

In another aspect of the present invention, the extract provides a water extract, a food composition for preventing or improving fine dust-stimulated respiratory diseases.

In another aspect of the present invention, the extract is extracted for 20 minutes to 10 hours at 50 ° C. to 150 ° C., providing a food composition for preventing or improving fine dust-stimulating respiratory diseases.

In another aspect of the present invention, the plants of the genus Citrus are pomelo (Citrus maxima), citron (Citrus medica), mandarin (Citrus reticulate), tangerine (Citrus trifoliate), golden persimmon (Citrus japonica), grapefruit (Citrus paradisi), citron (Citrus junos ) , Lemon (Citrus limon), Lime (Citrus aurantifolia), Tangerine (Citrus unshiu), Wenzhou tangerine (satsuma mandarin), and Sweet orange (Citrus sinensis), any one or more, to prevent or improve fine dust-stimulated respiratory diseases It provides a food composition for use.

In another aspect of the present invention, the plant of the genus Citrus is a fruit of the plant, providing a food composition for preventing or improving fine dust-stimulated respiratory diseases.

In another aspect of the present invention, the formulation of the food composition is not particularly limited, but may be formulated into, for example, tablets, granules, powders, liquids such as drinks, caramels, gels, bars, and the like. The food composition of each formulation can be selected and blended without difficulty by those skilled in the art according to the formulation or purpose of use other than the active ingredient, and synergistic effects can occur when applied simultaneously with other ingredients.

In the food composition according to another aspect of the present invention, the dosage determination of the active ingredient is within the level of those skilled in the art, and its daily dosage is, for example, 0.1 mg/kg/day to 5000 mg/kg/day, or more Specifically, it may be 50 mg/kg/day to 500 mg/kg/day, but is not limited thereto, and may vary depending on various factors such as the age, health condition, and complications of the subject to be administered.

The food composition according to another aspect of the present invention, for example, various foods such as chewing gum, caramel products, candies, frozen confectionery, and confectionery, beverage products such as soft drinks, mineral water, and alcoholic beverages, health products including vitamins and minerals, and the like It may be functional foods. In addition to the above, the food composition according to another aspect of the present invention is various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and salts thereof , alginic acid and its salts, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like. In addition, functional food compositions according to another aspect of the present invention may include natural fruit juice, fruit juice beverages, and fruit flesh for preparing vegetable beverages. These components may be used independently or in combination. The ratio of these additives is not so critical, but is generally included in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present specification.

Another aspect of the present invention provides a method for preparing the pharmaceutical composition or food composition.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for improving fine dust-stimulated respiratory diseases using an extract of the peel of a tangerine genus plant as an active ingredient and a method for preparing the same.

The plant of the genus Tangerine may be a fruit of Citrus junos, an evergreen broad-leaved shrub belonging to the Citrus family.

The pericarp of the Citron genus may include or exclude seeds of the citron, and may be at least one selected from the outer skin, mesocarp, and outer skin of the yuzu.

The extract may be a material extracted by selecting one of water, alcohol, and carbon dioxide. Preferably, 1,000 parts by weight of water based on 100 parts by weight of the peel of the tangerine genus plant is put in a reflux extraction equipment and the temperature is 0.5 to 100 ° C. It provides a manufacturing method that includes an 8-hour heating process, filtration, concentration, and drying steps.

One embodiment provides a composition for preventing and improving fine dust-stimulated respiratory diseases, including an extract of the peel of a tangerine genus plant as an active ingredient, and a method for preparing the same.

In one embodiment, the rind extract of the Citrus genus provides a composition for preventing and improving fine dust-stimulated respiratory diseases and a method for preparing the same, characterized in that obtained by hot water extraction of citron peel or citron peel.

In one embodiment, the tangerine plant rind extract contains naringin, and the naringin provides a composition for preventing and improving fine dust-stimulated respiratory diseases containing 0.34% or more by weight and a method for preparing the same.

In one embodiment, a composition for preventing and improving fine dust and fine dust-stimulated respiratory diseases by suppressing lung cell or bronchial inflammation, suppressing cell death or apoptosis of lung cells by the active ingredient and a method for producing the same.

In one embodiment, the extract is prepared by (A) preparing tangerine rind powder or gourd; And (B) adding a solvent to the peel powder or peel of the tangerine genus plant, followed by heating and extracting at 60 to 100 ° C. for 0.5 to 8 hours Provide a composition for preventing and improving fine dust-stimulated respiratory diseases prepared by an extraction method do.

In one embodiment, it provides a health functional food or pharmaceutical composition for preventing and improving fine dust-stimulating respiratory diseases containing the extract as an active ingredient and a method for preparing the same.

The present invention relates to a composition for preventing and improving fine dust-stimulated respiratory diseases using an extract of the peel of a tangerine genus plant as an active ingredient and a method for preparing the same.

The tangerine plant is preferably, but is not limited to, the fruit of Citrus junos, an evergreen broad-leaved shrub belonging to the Citrus family, and the tangerine plant pericarp may include or exclude seeds, and the outer skin, mesocarp or outer skin of the fruit It may be one or more selected from among.

The extract may be a material extracted by selecting one of water, alcohol, and carbon dioxide. Preferably, 1,000 parts by weight of water based on 100 parts by weight of the skin of the tangerine plant is heated in a reflux extraction equipment at 70 ° C. for 2 hours, and then filtered, It is preferable to proceed with the concentration and drying steps, but it is not limited thereto, and the process of heating for 0.5 to 8 hours at a temperature of 60 to 100 ° C., filtration, concentration, and drying may also be used without limitation.

The fruit of the citron tree can be used in the form of citron peel or citron peel, and the extract obtained from it has the characteristic of suppressing intracellular active oxygen.

The extract is characterized by suppressing the expression of inflammatory cytokines TNF-α and IL-1β in bronchial lavage fluid.

The extract is characterized by suppressing the expression of c-Caspase-9 and c-Caspase-3, which are cell death factors, in lung tissue.

The extract is characterized by suppressing the expression of IL-6, an inflammatory cytokine, in lung tissue.

Health functional food for preventing or improving fine dust-stimulated respiratory diseases containing citron (Citrus junos) blood or citron peel extract is manufactured as any one selected from beverages, pills, tablets, capsules, and powders, or other It can be prepared by adding it to food or ingredients of food, and can be suitably prepared according to conventional methods.

Examples of foods to which the Citrus junos blood or citron peel extract of the present invention can be added include meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gums, and ice creams. It may be in any one form selected from dairy products, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and includes all health foods in a conventional sense.

The health functional food includes various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, coloring agents and enhancers (cheese, chocolate, etc.), pectic acids and their salts, alkynic acids and their salts, organic acids, protective colloidal thickeners , pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, and the like. In addition, it may contain fruit flesh for the production of natural fruit juice and vegetable beverages. These components may be used independently or in combination.

The health functional food of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients. The natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used.

In addition, the present invention relates to a pharmaceutical composition for preventing or improving fine dust-stimulated respiratory diseases containing citron (Citrus junos) blood or citron peel extract as an active ingredient. The pharmaceutical composition of the present invention may further include a carrier, excipient or diluent in addition to the Citrus junos blood or citron peel extract. Carriers, excipients or diluents that may be preferably included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc and magnesium stearate, and the like, but are not limited thereto.

In addition, it can be administered in an oral or parenteral formulation according to a conventional method, and when formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, It is prepared by mixing gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral administration include suspensions, internal solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. can Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. In addition, vegetable oils such as propylene glycol, polyethylene glycol, olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base material for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.

Hereinafter, the present invention will be described in detail by production examples, examples and experimental examples. However, the following preparation examples, examples and experimental examples are only to illustrate the present invention, and the content of the present invention is not limited by the following examples.

Example

Examples 1 to 11. Mandarin orange peel extract preparation

The dried pericarp of the tangerine genus was pulverized to a particle size of 20 to 100 mesh, preferably 40 to 80 mesh. 10 L of purified water was added to 1 kg of the peel of the crushed tangerine genus plant, extracted at 70 ° C for 2 hours, and the filtered solution was concentrated under reduced pressure at 55 ° C and freeze-dried to obtain a moisture content of 10 to 30%, preferably 15 to 25%. An extract of the rind of the tangerine genus was obtained. Table 1 below shows the types of tangerine plant skins extracted and the extraction yield.

material name Export yield (%) Example 1 grapefruit blood 26.2 Example 2 cellar 15.4 Example 3 citron 29.4 Example 4 nojisil 27.5 Example 5 caracara orange 25.6 Example 6 ginseng skin 21.5 Example 7 dermis 22.5 Example 8 Lemon Pea 11.3 Example 9 Lime Pea 10.0 Example 10 hearing dog skin 12.4 Example 11 Kara scented skin 20.4

Examples 12 to 16. Preparation of citron extract according to extraction temperature

The dried citron peel was pulverized to a particle size of 20 to 100 mesh, preferably 40 to 80 mesh. 10 L of purified water was added to 1 kg of crushed citron peel, extracted at 60-100 ° C for 2 hours, and the filtered solution was concentrated under reduced pressure at 55 ° C and freeze-dried to a moisture content of 10-30%, preferably 15-25% A citron peel extract was obtained.

Table 2 below shows the extraction yield and naringin content of citron rinds by extraction temperature.

extraction temperature Extraction yield (%) Naringin ?t amount (mg/g) Naringin Yield (%) Example 12 60℃ 23.6 4.23 0.099 Example 13 70℃ 29.8 4.48 0.133 Example 14 80℃ 27.1 4.32 0.117 Example 15 90℃ 31.1 3.85 0.119 Example 16 100℃ 35.2 3.69 0.129

Examples 17 to 23. Preparation of citron extract according to extraction time

The dried citron peel was pulverized to a particle size of 20 to 100 mesh, preferably 40 to 80 mesh. 10 L of purified water was added to 1 kg of crushed citron peel, extracted at 70 ° C for 0.5 to 8 hours, and the filtered solution was concentrated under reduced pressure at 55 ° C and lyophilized to obtain a moisture content of 10 to 30%, preferably 15 to 25% A citron peel extract was obtained. Table 3 below shows the extraction yield and naringin content of citron rinds by extraction temperature.

Extraction time (h) Extraction yield (%) Naringin ?t amount (mg/g) Naringin Yield (%) Example 17 0.5 28.9 4.46 0.129 Example 18 One 28.0 4.61 0.129 Example 19 2 29.4 4.68 0.138 Example 20 3 21.7 4.55 0.099 Example 21 4 21.8 4.72 0.103 Example 22 6 27.2 4.59 0.125 Example 23 8 23.8 4.61 0.109

Example 24. Preparation of citron peel extract

To produce citron juice, 10 L of purified water was added to 1 kg of citron peels that had been juiced, extracted at 70 ° C for 2 hours, and the filtered liquid was concentrated under reduced pressure at 55 ° C and freeze-dried to have a moisture content of 10 to 30%. More specifically, a citron peel extract having a concentration of 15 to 25% was obtained. Table 4 below shows the extraction yield and naringin content of citron rind by extraction temperature.

material name Extraction yield (%) Naringin ?t amount (mg/g) Naringin Yield (%) Example 24 citron gourd 11.7 3.46 0.04

Test Example 1. Quantification of Naringin

Naringin is a flavone-7-o-glycoside that belongs to the flavonoids and is a naturally occurring substance in tangerine fruits. It is mainly found in grapefruit and citron and is one of the components that give the fruit bitterness. In particular, citrons do not have naringinase that decomposes naringin, so they still retain a bitter taste even when ripe, unlike other citrus fruits. Taking advantage of these characteristics, the naringin analysis of Examples 1 to 11 was performed using naringin as a standard material for standardization of the extract.

(1) HPLC analysis conditions

Column: Mightysil RP-18 GP 250-4.6 (5 uL)

Mobile phase: A solvent: 0.5% acetic acid in water, B solvent: acetonitrile

5-18% B (5 min; linear gradient), 18-18% B (30 min; isocratic), 18-100% B (35 min; linear gradient)

Flow rate: 1.0 mL/min

Detector: DAD detector, 280 nm

The results of quantification and yield of naringin of the peel extracts of tangerine genus plants prepared in Examples 1 to 11 are shown in Table 5 below.

material name Naringin ?t amount (mg/g) Naringin Yield (%) Example 1 grapefruit blood 12.31 0.322 Example 2 cellar 15.7 0.241 Example 3 citron 4.68 0.138 Example 4 nojisil 3.42 0.094 Example 5 caracara orange 0.32 0.008 Example 6 ginseng skin 0.28 0.006 Example 7 dermis 0.21 0.005 Example 8 Lemon Pea 0.46 0.005 Example 9 Lime Pea 0.38 0.004 Example 10 hearing dog skin 0.18 0.002 Example 11 Kara scented skin 0.12 0.002

Test Example 2. Antioxidant activity

(1) ABTS radical scavenging activity analysis

ABTS radical scavenging activity was analyzed for Examples 1, 2, 3, 4, and 7 in order to investigate the antioxidant effect of the peel extract of Tangerine. 1 mM AAPH (2,2'-Azobis(2-amidinopropane) dihydrochloride) and 2.5 mM ABTS (2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) reagent were dissolved in 1 L of PBS. After stabilizing the radical by heating at 70 ° C for 1 hour, cooling at room temperature for 20 to 30 minutes, then diluting the ABTS radical solution with PBS so that the absorbance value is 0.65 at a wavelength of 734 nm, and then adding 1.47 mL of the ABTS radical solution. 0.03 mL (1 mg/mL) of the sample was mixed and reacted in the dark at room temperature for 10 minutes, and then the absorbance at 734 nm was measured. The results are shown in (A) of FIG.

(2) DPPH radical scavenging activity analysis

DPPH radical scavenging activity was analyzed for Examples 1, 2, 3, 4, and 7 in order to investigate the antioxidant effect of the peel extract of Tangerine. DPPH radical scavenging activity was determined by dissolving 2,2-Diphenyl-1-picryl-hydrazyl reagent in EtOH to prepare a 0.1 mM DPPH radical solution, and then diluting the DPPH radical solution in EtOH to an absorbance value of 0.7 at 517 nm. Then, 0.05 mL (1 mg/mL) of the sample was added to 1.45 mL of DPPH radical solution, followed by dark reaction for 30 minutes, and absorbance at 517 nm was measured. The measured absorbance value was converted from a standard curve using vitamin C and expressed as VCEAC (Vitamin C Equivalent Antioxidant Capacity). The results are shown in (B) of FIG.

As shown in (A) and (B) of Figure 1, it was difficult to confirm the correlation between the naringin concentration and the antioxidant effect. This can be judged to be an inherent effect of not only Naringin but also other constituents depending on the type of tangerine fruit.

Test Example 3. Lung cell protective effect

MTT assay was performed to confirm the toxicity of fine dust to lung cells. 100 μL (1 × 10⁴ cell) of lung cells (NCI-H460) was dispensed into a 96-well plate, cultured for 24 hours, and Examples 1, 2, 3, 4, and 7 were added at a concentration of 0.8 mg/mL. After replacement with cell culture medium, 0.4 mg/mL of PM10 was treated for 24 hours to induce oxidative stress. Thereafter, 10 μL of MTT reagent at a concentration of 5 mg/mL was added to each well, treated for 3 hours, and the resulting formazan was dissolved in DMSO to measure absorbance at 595 nm.

As shown in Figure 2, it can be confirmed that the citron and grapefruit peel extracts significantly inhibit lung cell death caused by fine dust to a degree similar to that of naringin used as a positive control.

Test Example 4. Analysis of reactive oxygen species (ROS) removal in lung cells

In order to check the amount of reactive oxygen species (ROS) produced in lung cells by PM10 administration, 100 μL (3×10⁴ cells) of lung cells (NCI-H460) were dispensed into a 96-well black plate and cultured for 24 hours. After that, the medium was removed, and 75 μL (0.8 mg/mL) of the sample medium and 25 μL of 100 μM DCFH-DA (2'-7'dichlorofluorescin diacetate) were dispensed and cultured for 1 hour. After 1 hour, oxidative stress was induced by treatment with 600 uM AAPH (2,2′-Azobis(2-methylpropionamidine) dihydrochloride), and extinction 485 nm, emission was observed using a fluorometer for 1 hour at 5-minute intervals immediately after treatment. Fluorescence was measured under the condition of 535 nm.

As shown in FIG. 3, it was confirmed that intracellular reactive oxygen species generated by AAPH can be significantly reduced in citron and grapefruit peel extracts. This means that the respiratory antioxidant mechanism is reduced by fine dust, and oxidative stress is directly applied to epithelial cells, which increases the expression of NF-kB, a transcription factor, and increases inflammatory cytokines and enzymes, thereby increasing lung cell inflammation and cancer. According to previously known information (Yang W, Omaye ST (2009) Air pollutants, oxidative stress and human health. Mutat Res 674:45-54), oxidative stress caused by fine dust can be effectively reduced, thereby reducing the respiratory tract stimulated by fine dust. It suggests that the disease can be improved.

Test Example 5. Evaluation of Lung Damage Inhibiting Effect in Experimental Animals

(1) Experimental animals and test substances

Test animals: 7-week-old BALB/c male mice

Breeding environment: 23±1℃ (temperature), 55±1% (humidity), light/dark cycle 12 hours

Test group: Normal group (NC), control group (CON), positive control group (NAR; Naringin), Example 3 100 mg/kg (CPL), Example 3 200 mg/kg (CPH), Example 24 100 mg/kg kg (CBL), Example 24 200 mg/kg (CBH)

(2) Treatment of fine dust-induced lung damage and experimental substances

After a 7-day adaptation period, BALB/c mice were orally administered with the test substances in a volume of 200 μL once daily for 7 days, and 30 minutes after the last oral administration, PM10 was administered at 10 mg/kg B.W. After intranasal administration of 40 μL each of 20 μL to both nasal cavities at the same concentration, the cells were dissected after 6 hours.

(3) Confirmation of lung damage and protective effect

To measure the degree of lung damage, the lung tissue was fixed in 10% neutral buffered formalin for 24 hours, dehydrated using graded alcohol, embedded in paraffin, and a block was made. Then, using a microtome, a 3 μm thick tissue section was obtained and hematoxylinin & eosin (H&E) reagent. The stained tissue slide was wetted with mounting solution, a cover-slide was attached, observed under an optical microscope at × 100 magnification, and photographed with a digital camera.

As shown in Figure 4, in the control group induced by fine dust (PM10), inflammatory cell infiltration in lung tissue was observed more than the normal group, and in the positive control group (naringin) and Experimental Example 3 and Experimental Example 24 administration groups, fine dust Inflammatory cell infiltration in stimulated lung cells was less observed, confirming that the lung cell inflammation stimulated by fine dust can be effectively reduced through the ingestion of Experimental Examples 3 and 24.

(4) Analysis of inflammatory cytokine expression

Enzyme-Linked Immunosorbent Assay (ELISA) was performed to measure the expression level of inflammatory cytokines (TNF-α, IL-1β) in bronchoalveolar lavage fluid (BALF). Specifically, 50 μL of diluent assay buffer and 50 μL of BALF solution were added per well to a 96-well plate coated with Capture antibody, reacted for 2 hours, washed 4 times using wash buffer, and biotin-conjugate antibody reagent was placed in each well. 100 μL each and reacted at room temperature for 2 hours. Thereafter, after washing four times, each well was treated with substrate solution and allowed to react in the dark at room temperature for 30 minutes, and then treated with 100 μL of stop solution to terminate the reaction, and the absorbance was measured at 540 nm.

As shown in FIG. 5, it was confirmed that a significant reduction effect of TNF-α, one of the acutely occurring inflammatory cytokines, was confirmed in the test animals administered with Examples 3 and 24. This is a result of supporting the fact that the expression of NF-kB in the respiratory tract by fine dust increases, which increases the expression of inflammatory cytokines, and the expression of TNF-α is increased by fine dust, which is presented in the present invention It is suggested that the compositions of Examples 3 to 24 can effectively inhibit NF-kB expression and effectively inhibit the expression of various inflammatory cytokines.

(5) Analysis of apoptosis factors and inflammatory cytokines in lung tissue

Lung tissues were excised, rapidly frozen in liquid nitrogen, and stored frozen (-70 °C). For protein extraction in tissue, 0.5 mL of lysis buffer and three 3 mm glass beads were added per 100 mg of lung tissue, and tissue disruption was performed using a bead beater. After standing at 4℃ for 1 hour, centrifugation was performed at 4℃ and 16,000 × g for 15 minutes using a rotator to separate the protein between the fat layer and the pellet, and the protein was quantified using the Bradford method. As the standard solution, bovine serum albumin (BSA) was set at a concentration of 0 to 2 mg/mL, and 2 μL of the sample and 200 μL of bradford solution were put in a 96-well plate, and the absorbance was measured at a wavelength of 595 nm, and then the BSA standard curve was used. The concentration of protein was calculated.

Then, the proteins are separated by size by electrophoresis using SDS-PAGE, transferred to a NC (Nitrocellulose) membrane, and then attached with a specific primary antibody for each at 4 ° C for 16 hours, followed by attachment of a secondary antibody for 1 hour at room temperature. Protein expression levels were measured.

Enzyme-Linked Immunosorbent Assay (ELISA) was performed to measure IL-6, an inflammatory cytokine in lung tissue. Specifically, 50 μL of diluent assay buffer and 50 μL of BALF solution were added per well to a 96-well plate coated with Capture antibody, reacted for 2 hours, washed 4 times using wash buffer, and biotin-conjugate antibody reagent was placed in each well. 100 μL each and reacted at room temperature for 2 hours. Thereafter, after washing four times, each well was treated with substrate solution and allowed to react in the dark at room temperature for 30 minutes. Then, 100 μL of stop solution was treated to terminate the reaction, and absorbance was measured at 540 nm.

The experimental results were the same as those in FIGS. 6 to 7.

As shown in FIG. 6, it was confirmed that the expression level of c-Caspase-3, an apoptosis-related protein, decreased in the test groups administered with Examples 3 and 24. It was also confirmed that the expression level of c-Caspase-9, an upper signaling protein of 3, also decreased.

The expression pattern of IL-6, an inflammatory cytokine in lung cells, is shown in FIG. 7 . IL-6 is an inflammatory cytokine generated in the early stage of an inflammatory response and is known to promote an immune response by being secreted from various cells such as T lymphocytes and macrophages. In particular, it plays a role in promoting various inflammatory reactions that lead to tissue damage caused by inflammation. Example 24 In the 100 mg/kg administration group, it was confirmed that the expression level of IL-6 in lung cells stimulated by fine dust was significantly reduced. suggests that it can be improved.

formulation example

Formulation examples of the composition according to one aspect of the present invention will be described below, but can also be applied to various other formulations, which are only intended to be specifically described, not intended to limit the present specification.

[Formulation Example 1] Soft capsule

8 mg of the extract of Example, 9 mg of vitamin E, 9 mg of vitamin C, 2 mg of palm oil, 8 mg of hydrogenated vegetable oil, 4 mg of yellow wax, and 9 mg of lecithin were mixed and mixed according to a conventional method to prepare a soft capsule filling solution. Soft capsules are prepared by filling 400 mg per capsule. In addition to the above, a soft capsule sheet containing 400 mg of the composition according to the present specification is prepared by preparing a soft capsule sheet at a ratio of 66 parts by weight of gelatin, 24 parts by weight of glycerin, and 10 parts by weight of sorbitol solution, and filling the filling solution.

[Formulation Example 2] Tablets

8 mg of the extract of Example, 9 mg of vitamin E, 9 mg of vitamin C, 200 mg of galactooligosaccharide, 60 mg of lactose, and 140 mg of maltose were mixed and granulated using a fluid bed dryer, and then 6 mg of sugar ester was added. Tablets were prepared by compressing 500 mg of these compositions in a conventional manner.

[Formulation Example 3] Drinking agent

After mixing 8mg of the extract of Example, 9mg of vitamin E, 9mg of vitamin C, 10g of glucose, 0.6g of citric acid, and 25g of liquid oligosaccharide, 300㎖ of purified water was added to fill each bottle to 200㎖. After filling the bottle, sterilize at 130 ° C. for 4 to 5 seconds to prepare a drink.

[Formulation Example 4] Granules

8 mg of the extract of Example, 9 mg of vitamin E, 9 mg of vitamin C, 250 mg of anhydrous crystalline glucose and 550 mg of starch were mixed, molded into granules using a fluid bed granulator, and then filled into bags to prepare granules.

[Formulation Example 5] Injection

Injections were prepared by a conventional method according to the compositions shown in Table 6 below.

formulation content Extracts of Examples 10-50mg Sterile Distilled Water for Injection Appropriate amount pH modifier Appropriate amount

[Formulation Example 6] Health drink

A health drink was prepared in a conventional manner according to the composition shown in Table 7 below.

formulation content Extracts of Examples 1000 mg citric acid 1000 mg oligosaccharide 100 grams taurine 1g Purified water balance

After mixing the above components according to a conventional health drink manufacturing method, stirring and heating at 85 ° C. for about 1 hour, the resulting solution is filtered and sterilized.

Above, the present invention has been described through examples, experimental examples, and manufacturing examples, but those skilled in the art to which the present invention pertains can implement the present invention in other specific forms without changing its technical spirit or essential features. You will understand that you can. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (12)

In the pharmaceutical composition for preventing or treating respiratory diseases,
It contains extracts of plants of the genus Citrus as active ingredients,
The extract is an extract of water, alcohol having 1 to 5 carbon atoms or an aqueous solution of alcohol having 1 to 5 carbon atoms, and the extract is extracted at 50 ° C to 150 ° C for 20 minutes to 10 hours,
The respiratory disease is caused by fine dust and is a respiratory disease caused by the expression of inflammatory factors TNF-α, IL-6 and IL-1β in lung cells and bronchial tissues,
A pharmaceutical composition for preventing or treating respiratory diseases.
delete delete delete According to claim 1,
Plants of the genus Citrus include pomelo (Citrus maxima), citron (Citrus medica), mandarin (Citrus reticulate), tangerine (Citrus trifoliate), golden persimmon (Citrus japonica), grapefruit (Citrus paradisi), citron (Citrus junos ) , lemon (Citrus limon), lime (Citrus aurantifolia), tangerine (Citrus unshiu), mandarin orange (satsuma mandarin), and sweet orange (Citrus sinensis), any one or more of, a pharmaceutical composition for preventing or treating fine dust-stimulated respiratory diseases.
According to claim 1,
The plant of the genus Citrus is a fruit of a plant, a pharmaceutical composition for preventing or treating fine dust-stimulated respiratory diseases.
In the food composition for preventing or improving respiratory diseases,
Contains extracts of plants of the genus Citrus as active ingredients,
The extract is an extract of water, alcohol having 1 to 5 carbon atoms or an aqueous solution of alcohol having 1 to 5 carbon atoms, and the extract is extracted at 50 ° C to 150 ° C for 20 minutes to 10 hours,
The respiratory disease is caused by fine dust and is a respiratory disease caused by the expression of inflammatory factors TNF-α, IL-6 and IL-1β in lung cells and bronchial tissues,
A food composition for preventing or improving respiratory diseases.
delete delete delete According to claim 7,
Plants of the genus Citrus include pomelo (Citrus maxima), citron (Citrus medica), mandarin (Citrus reticulate), tangerine (Citrus trifoliate), golden persimmon (Citrus japonica), grapefruit (Citrus paradisi), citron (Citrus junos ) , lemon (Citrus limon), lime (Citrus aurantifolia), tangerine (Citrus unshiu), satsuma mandarin, and sweet orange (Citrus sinensis), any one or more of, a food composition for preventing or improving fine dust-stimulated respiratory diseases.
According to claim 7,
The plant of the genus Citrus is a fruit of a plant, a food composition for preventing or improving fine dust-stimulating respiratory diseases.

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