KR20220046810A - 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 alleviating fine dust-stimulated respiratory diseases, containing an extract of the peel of a citrus plant as an active ingredient, and a method for manufacturing the same. The present invention can be effectively used to prevent and alleviate respiratory diseases stimulated by fine dust by reducing free radicals (ROS) in tissues and cells.

Description

Method for manufacturing a composition for improving fine dust stimulation respiratory disease using the peel of a citrus plant {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, and a method for manufacturing the same, using an extract of the fruit skin of a citrus plant as an active ingredient, and more particularly, to the prevention of fine dust-stimulated respiratory diseases obtained by hot water extraction of citron skin or citron meal And it relates to a composition for improvement and a method for preparing the same.

Since the 1960s, Korea has undergone rapid industrialization and urban concentration due to the promotion of economic development, the development and dissemination of science and technology, and industrialization. The situation is serious. As data to support this, there were reports that Korea's air pollution level is the worst among the Organization for Economic Cooperation and Development (OECD) countries and the 26th most severe in the world (2019 World Air Quality Report, IQ Air (Switzerland)).

According to the report, 21 out of 30 air polluting cities are located in India and the rest are located in Asia. Like Korea, China, which is adjacent to Korea, has rapid industrialization, industrialization, and dependence on coal as high as 70% or more. Due to this, fine dust warnings and warnings are also increasing in Korea.

Fine dust is caused by combustion of coal fuel, exhaust gas from automobiles, road dust, etc., but mainly contains very complex components such as sulfur oxides, lead, ozone, carbon monoxide, and nitrogen oxides and is floating in the atmosphere.

Although the composition and size of fine dust are very diverse, it can be divided into ultra-fine dust with a diameter of 2.5 um or less called PM2.5 and fine dust with a diameter of 10 uM or less called PM10. It has been reported to adversely affect organs and sites.

In particular, the effect on the respiratory tract that comes into contact with fine dust for the first time is mainly caused by an inflammatory reaction in the bronchioles. These stimuli are well known as the cause of chronic bronchitis, asthma, chronic obstructive respiratory disease, etc.

In addition to this, lung diseases caused by fine dust are well known for lung damage and pulmonary edema.

Moreover, there is a report that fine dust interferes with the self-defense mechanism of lung cells, reducing or losing the ability to cope with various bacteria invading lung cells, which in turn causes various respiratory infections.

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

As such, it is predicted that the social cost loss will reach 10 trillion won and 20 trillion won in 2060 due to the threat to public health caused by air pollution, especially fine dust (Bae Jeong-hwan, Professor of Economics, Chonnam National University, 2017). Efforts and investments to improve the environment are continuously being made worldwide, and there is a need to develop foods and drugs 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 few side effects and excellent functions by using beneficial ingredients of natural products.

Korean Patent Publication No. 10-2019-0127583 Korean 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, and a method for manufacturing the same, comprising an extract of the peel of a tangerine plant as an active ingredient.

In order to solve the above problems, the present invention provides means for solving the problems as follows.

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 is water, an alcohol having 1 to 5 carbon atoms or an aqueous solution of alcohol having 1 to 5 carbon atoms is extracted as an extraction solvent, it provides a pharmaceutical composition for preventing or treating fine dust stimulation respiratory disease .

In one aspect of the present invention, the extract is a water extract, provides a pharmaceutical composition for preventing or treating fine dust stimulation respiratory disease.

In one aspect of the present invention, the extract is extracted for 20 minutes to 10 hours at 50 ℃ to 150 ℃, provides a pharmaceutical composition for preventing or treating fine dust stimulation respiratory disease.

In one aspect of the present invention, the citrus genus plants are pomelo (Citrus maxima), citrus (Citrus medica), mandarin (Citrus reticulate), tangja (Citrus trifoliate), geumgam (Citrus japonica), grapefruit (Citrus paradisi), citron (Citrus junos ) , lemon (Citrus limon), lime (Citrus aurantifolia), tangerine (Citrus unshiu), wenzhou citrus (satsuma mandarin), and sweet orange (Citrus sinensis), any one or more of, prevention or treatment of fine dust-stimulating respiratory diseases A pharmaceutical composition is provided.

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

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 is water, an alcohol having 1 to 5 carbon atoms or an aqueous solution of alcohol having 1 to 5 carbon atoms is extracted as an extraction solvent, it provides a food composition for preventing or improving fine dust stimulation respiratory disease .

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

In another aspect of the present invention, the extract is extracted for 20 minutes to 10 hours at 50 ℃ to 150 ℃, provides a food composition for preventing or improving fine dust stimulatory respiratory disease.

In another aspect of the present invention, the citrus genus plants are pomelo (Citrus maxima), citrus (Citrus medica), mandarin (Citrus reticulate), tangja (Citrus trifoliate), geumgam (Citrus japonica), grapefruit (Citrus paradisi), citron (Citrus junos ) , lemon (Citrus limon), lime (Citrus aurantifolia), tangerine (Citrus unshiu), satsuma mandarin (satsuma mandarin), and sweet orange (Citrus sinensis), any one or more of, prevention or improvement of fine dust-stimulating respiratory diseases It provides a food composition for use.

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

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 the peel of a citrus plant, preferably, citron peel or citron gourd as an active ingredient, and a method for manufacturing the same. Inhibits the expression of inflammatory factors TNF-α, IL-6, IL-1β, and c-Caspsae, a signaling mechanism of cell death or apoptosis in lung cells, and inhibits the expression of reactive oxygen species (ROS) in tissues and cells. ) 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 the result of confirming the antioxidant effect of the peel extract of citrus plants. Figure 1 (A) is ABTS, (B) is the DPPH result.
Figure 2 is the result of confirming the cytoprotective effect on the lung cell death induced by fine dust of the extract of tangerine plant peel.
3 is a result confirming the inhibitory effect of ROS generation due to intracellular oxidative stress of a tangerine peel extract.
4 is a result confirming the preventive effect of citron skin and citron gourd extract on lung damage caused by fine dust (PM10) in experimental animals.
5 is a result confirming the effect of inhibiting the expression of inflammatory cytokines (TNF-α, IL-1β) by citron skin and citron extract in bronchial lavage (BALF) of experimental animals.
6 is a result confirming the effect of inhibiting the expression of apoptosis factors (c-Caspase-9, c-Caspase-3) by citron skin and citron extract in the lung tissue of an experimental animal.
7 is a result confirming the inhibitory effect of inflammatory cytokine (IL-6) expression by citron skin and citron extract in lung tissue of experimental animals.

Unless otherwise specified, all numbers, values, and/or expressions expressing ingredients, reaction conditions, and amounts of ingredients used herein refer to a variety of measures that may occur in obtaining such values, among others, in which such numbers are inherently different. Since they are approximations reflecting uncertainty, it should be understood as being modified by the term “about” in all cases. Also, where the disclosure discloses numerical ranges, such ranges are continuous and inclusive of all values from the minimum to the maximum inclusive of the range, unless otherwise indicated. Furthermore, when such ranges refer to integers, all integers inclusive from the minimum to the maximum inclusive are included, unless otherwise indicated.

In this specification, when a range is described for a variable, the variable will be understood to include all values within the stated range including the stated endpoints of the range. For example, a range of “5 to 10” includes the values of 5, 6, 7, 8, 9, and 10, as well as any subranges such as 6 to 10, 7 to 10, 6 to 9, 7 to 9, etc. It will be understood to include any value between integers that are appropriate for the scope of the recited 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, ranges from "10% to 30%" include values of 10%, 11%, 12%, 13%, etc. and all integers up to and including 30%, as well as 10% to 15%, 12% to It will be understood to include any subranges such as 18%, 20% to 30%, etc., as well as any value between reasonable integers within the scope of the recited ranges, 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 is water, an alcohol having 1 to 5 carbon atoms or an aqueous solution of alcohol having 1 to 5 carbon atoms is extracted as an extraction solvent, it provides a pharmaceutical composition for preventing or treating fine dust stimulation respiratory disease .

In one aspect of the present invention, the extract is a water extract, provides a pharmaceutical composition for preventing or treating fine dust stimulation respiratory disease.

In one aspect of the present invention, the extract is extracted for 20 minutes to 10 hours at 50 ℃ to 150 ℃, provides a pharmaceutical composition for preventing or treating fine dust stimulation respiratory disease.

In one aspect of the present invention, the citrus genus plants are pomelo (Citrus maxima), citrus (Citrus medica), mandarin (Citrus reticulate), tangja (Citrus trifoliate), geumgam (Citrus japonica), grapefruit (Citrus paradisi), citron (Citrus junos ) , lemon (Citrus limon), lime (Citrus aurantifolia), tangerine (Citrus unshiu), wenzhou citrus (satsuma mandarin), and sweet orange (Citrus sinensis), any one or more of, prevention or treatment of fine dust-stimulating respiratory diseases A pharmaceutical composition is provided.

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

The pharmaceutical composition according to one aspect of the present invention may be in various oral or parenteral formulations. In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, soft or hard capsules, etc., and such solid preparations include at least one excipient in one or more compounds, for example, starch, calcium carbonate, sucrose Alternatively, it is prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can 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, 1 to several times. It can be administered in divided doses. The dosage for a specific patient may vary depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, severity of disease, and the like.

The pharmaceutical composition according to one aspect of the present invention is a powder, granules, tablets, soft or hard capsules, suspensions, emulsions, syrups, drinks, oral dosage forms such as 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, and specifically formulated in the form of injections or external preparations for skin.

The pharmaceutical composition according to one aspect of the present invention may be administered to mammals such as rats, mice, livestock, and humans by various routes such as parenteral and oral administration, and all methods of administration can be expected, for example, , oral, 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 by 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 an external preparation for the skin by 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 is water, an alcohol having 1 to 5 carbon atoms or an aqueous solution of alcohol having 1 to 5 carbon atoms is extracted as an extraction solvent, it provides a food composition for preventing or improving fine dust stimulation respiratory disease .

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

In another aspect of the present invention, the extract is extracted for 20 minutes to 10 hours at 50 ℃ to 150 ℃, provides a food composition for preventing or improving fine dust stimulatory respiratory disease.

In another aspect of the present invention, the citrus genus plants are pomelo (Citrus maxima), citrus (Citrus medica), mandarin (Citrus reticulate), tangja (Citrus trifoliate), geumgam (Citrus japonica), grapefruit (Citrus paradisi), citron (Citrus junos ) , lemon (Citrus limon), lime (Citrus aurantifolia), tangerine (Citrus unshiu), satsuma mandarin (satsuma mandarin), and sweet orange (Citrus sinensis), any one or more of, prevention or improvement of fine dust-stimulating respiratory diseases It provides a food composition for use.

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

In another aspect of the present invention, the formulation of the food composition is not particularly limited, but may be formulated as, for example, tablets, granules, powders, liquids such as drinks, caramel, gels, bars, and the like. In addition to the active ingredient, the food composition of each dosage form can be appropriately selected and formulated by those skilled in the art without difficulty depending on the dosage form or purpose of use in addition to the active ingredient, and a synergistic effect may occur when applied simultaneously with other raw materials.

In the food composition according to another aspect of the present invention, the determination of the dosage of the active ingredient is within the level of those skilled in the art, and the daily dosage thereof is, for example, 0.1 mg/kg/day to 5000 mg/kg/day, 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 status, and complications of the subject to be administered.

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

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, and a method for preparing the same, comprising an extract of the peel of a citrus plant as an active ingredient.

The citrus plant may be a fruit of Citrus junos, which is an evergreen broad-leaved shrub belonging to the Naeun family.

The pericarp of the citrus plant may include or exclude the seeds of citron, and may be one or more selected from the outer skin of citron, the inner skin and the outer skin.

The extract may be a material extracted by selecting one of water, alcohol, and carbon dioxide, and preferably, based on 100 parts by weight of the peel of the citrus plant, 1,000 parts by weight of water is placed in a reflux extraction equipment and 0.5 to 100 parts by weight at a temperature of 60 to 100 ° C. It provides a manufacturing method that includes heating for 8 hours, filtration, concentration, and drying steps.

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

In one embodiment, the citrus fruit peel extract provides a composition for preventing and improving fine dust-stimulated respiratory diseases, characterized in that it is obtained by hot-water extraction of citron skin or citron gourd, and a method for manufacturing the same.

In one embodiment, the citrus fruit peel extract contains naringin, and the naringin contains 0.34% or more by weight to provide a composition for preventing and improving fine dust-stimulated respiratory diseases, and a method for manufacturing the same.

In one embodiment, the composition for preventing and improving fine dust and fine dust stimulation respiratory diseases caused by the inhibition of inflammation of lung cells or bronchial tubes, inhibition of cell death of lung cells, or inhibition of apoptosis by the active ingredient And it provides a manufacturing method thereof.

In one embodiment, the extract comprises the steps of (A) preparing a citrus fruit peel powder or gourd; And (B) after adding a solvent to the citrus plant peel powder or gourd, heat extraction at 60 to 100° C. for 0.5 to 8 hours Provides a composition for preventing and improving fine dust-stimulated respiratory diseases prepared by an extraction method do.

In one embodiment, there is provided a health functional food or pharmaceutical composition for preventing and improving fine dust-stimulated respiratory diseases containing the extract as an active ingredient, and a method for manufacturing the same.

The present invention relates to a composition for preventing and improving fine dust-stimulated respiratory diseases, comprising an extract of the peel of a citrus plant as an active ingredient, and a method for manufacturing the same.

Preferably, the citrus plant is a fruit of Citrus junos, an evergreen broad-leaved shrub belonging to the Suwon family, but is not limited thereto, and the pericarp of the citrus plant may include or exclude seeds. 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, and preferably, 1,000 parts by weight of water based on 100 parts by weight of the pericarp of the citrus plant is heated at 70° C. in a reflux extraction equipment for 2 hours, followed by filtration; It is preferable to proceed with the concentration and drying steps, but the process of heating at a temperature of 60 to 100° C. for 0.5 to 8 hours, filtration, concentration, and drying may also be freely used, without being limited thereto.

The fruit of the citron tree can be used in the form of citron skin or citron gourd, and the extract obtained therefrom has a characteristic of inhibiting intracellular free radicals.

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

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

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

Health functional food for the prevention or improvement of fine dust-stimulating respiratory diseases containing Citrus junos blood or citron extract is manufactured with any one selected from beverages, pills, tablets, capsules, and powders, or It may be prepared by adding it to food or food ingredients, and may be appropriately prepared according to a conventional method.

Examples of foods to which the Citrus junos blood or citron extract of the present invention can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, and ice cream. It may be in any one form selected from dairy products, various soups, beverages, teas, 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 flavorants, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alkynic acid and its salts, organic acids, protective colloidal thickeners , a pH adjuster, a stabilizer, a preservative, glycerin, alcohol, a carbonation agent used in carbonated beverages, and the like. In addition, it may contain the pulp for the production of natural fruit juices 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 tau martin 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 Citrus junos blood or citron 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 extract. Preferably, carriers, excipients or diluents that may be 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.

In addition, it may be administered in oral or parenteral formulations according to a conventional method, and in the case of formulation, usually used fillers, extenders, binders, wetting agents, disintegrants, diluents such as surfactants or excipients are used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the composition, for example, 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, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. can Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. In addition, as a non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. may be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

Hereinafter, the present invention will be described in detail with reference to Preparation Examples, Examples and Experimental Examples. However, the following Preparation Examples, Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples.

Example

Examples 1 to 11. Preparation of citrus fruit peel extract

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

Material name Export yield (%) Example 1 grapefruit 26.2 Example 2 branch 15.4 Example 3 Citron 29.4 Example 4 Noh Ji-sil 27.5 Example 5 Caracara Orange 25.6 Example 6 Jinjihyangpi 21.5 Example 7 dermis 22.5 Example 8 Lemon Peel 11.3 Example 9 Lime Pea 10.0 Example 10 blue dog skin 12.4 Example 11 cara incense peel 20.4

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

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

In Table 2 below, the extraction yield and naringin content of citron peel for each extraction temperature were indicated.

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. After adding 10 L of purified water to 1 kg of crushed citron peel and extracting at 70° C. for 0.5 to 8 hours, the filtered solution is 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. In Table 3 below, the extraction yield and naringin content of citron peel for each extraction temperature were indicated.

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 Yuja Bak Extract

To prepare citron juice, 10 L of purified water is added to 1 kg of citron juice that has been extracted and extracted for 2 hours at 70 ° C. A citron extract at 15-25% was obtained. Table 4 below shows the extraction yield and naringin content of citron peel for each extraction temperature.

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

Test Example 1. Quantification of Naringin

Naringin is a flavone-7-o-glycoside that belongs to a flavonoid and is a naturally occurring substance in citrus fruits. It is mainly found in grapefruit and citron and is one of the components that give the fruit a bitter taste. In particular, citron does not contain naringinase that breaks down naringin, so it still has a bitter taste, unlike other citrus fruits, even when ripe. To standardize the extract by utilizing these characteristics, naringin was used as a standard material and the naringin analysis of Examples 1 to 11 was performed.

(One) 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

Table 5 below shows the naringin quantitative and yield results of the citrus fruit peel extracts prepared in Examples 1 to 11.

Material name Naringin ?t amount (mg/g) Naringin Yield (%) Example 1 grapefruit 12.31 0.322 Example 2 branch 15.7 0.241 Example 3 Citron 4.68 0.138 Example 4 Noh Ji-sil 3.42 0.094 Example 5 Caracara Orange 0.32 0.008 Example 6 Jinjihyangpi 0.28 0.006 Example 7 dermis 0.21 0.005 Example 8 Lemon Peel 0.46 0.005 Example 9 Lime Pea 0.38 0.004 Example 10 blue dog skin 0.18 0.002 Example 11 cara incense peel 0.12 0.002

Test Example 2. Antioxidant activity

(1) Analysis of ABTS radical scavenging ability

ABTS radical scavenging ability was analyzed for Examples 1, 2, 3, 4, and 7 in order to investigate the antioxidant effect of the tangerine peel extract. Dissolve 1 mM AAPH (2,2'-Azobis(2-amidinopropane) dihydrochloride) and 2.5 mM ABTS (2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) reagents in 1 L of PBS. The radicals were stabilized by heating at 70° C., and then cooled for 20 to 30 minutes at room temperature. After that, the ABTS radical solution was diluted with PBS so that the absorbance value at a wavelength of 734 nm was 0.65, and then 1.47 mL of the ABTS radical solution 0.03 mL (1 mg/mL) of the sample was mixed with , followed by dark reaction at room temperature for 10 minutes, and then absorbance at 734 nm was measured. The results are shown in Fig. 1 (A).

(2) DPPH radical scavenging ability analysis

DPPH radical scavenging ability was analyzed for Examples 1, 2, 3, 4, and 7 to investigate the antioxidant effect of the tangerine peel extract. The DPPH radical scavenging ability was obtained 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 so that the absorbance value at 517 nm was 0.7. Then, 0.05 mL (1 mg/mL) of the sample was added to 1.45 mL of the DPPH radical solution, and after dark reaction for 30 minutes, the 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 FIG. 1(B).

As shown in (A) and (B) of Figure 1, it was difficult to confirm the correlation between the concentration of naringin and the antioxidant effect. This can be judged to be an inherent effect of other constituents according to the type of tangerine fruit as well as naringin.

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 cells) of lung cells (NCI-H460) were dispensed into 96 well plates and cultured for 24 hours, and Examples 1, 2, 3, 4, and 7 were added at a concentration of 0.8 mg/mL. After replacing the cell culture medium, oxidative stress was induced by treatment with 0.4 mg/mL of PM10 for 24 hours. Then, 10 μL of MTT reagent at a concentration of 5 mg/mL was added to each well, treated for 3 hours, the culture medium was removed, and the formed formazan was dissolved in DMSO to measure absorbance at 595 nm.

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

Test Example 4. Analysis of Lung Cellular Reactive Oxygen (ROS) Removal Ability

To check the amount of reactive oxygen species (ROS) production 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. Then, 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 incubated for 1 hour. After 1 hour, 600 uM of AAPH (2,2′-Azobis(2-methylpropionamidine) dihydrochloride) was treated to induce oxidative stress, and immediately after treatment, using a fluorometer at 5-minute intervals for 1 hour, extinction 485 nm, Emission. Fluorescence was measured at 535 nm.

As shown in Figure 3, it was confirmed that the intracellular free radicals generated by AAPH in the extracts of citron and grapefruit could be significantly reduced. This is because the respiratory antioxidant mechanism is reduced by fine dust and oxidative stress is directly applied to epithelial cells, thereby increasing the expression of NF-kB, a transcription factor, and increasing inflammatory cytokines and enzymes to increase lung cell inflammation and cancer. Respiratory stimuli stimulated by fine dust by effectively reducing oxidative stress caused by fine dust according to previously known contents (Yang W, Omaye ST (2009) Air pollutants, oxidative stress and human health. Mutat Res 674:45-54) It suggests that the disease can be ameliorated.

Test Example 5. Evaluation of Lung Injury Inhibition Effect in Experimental Animals

(1) Experimental animals and test substances

Test animal: 7-week-old BALB/c male mouse

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 (CBL), Example 24 200 mg/kg (CBH)

(2) Fine dust-induced lung damage and treatment of experimental substances

BALB/c mice, which had undergone a 7-day adaptation period, were orally administered with test substances in a volume of 200 μL once daily for 7 days, and PM10 was administered at 10 mg/kg B.W. After intranasal administration of 20 μL of 40 μL to both nasal passages as a concentration, dissection was performed 6 hours later.

(3) check the 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, and embedded in paraffin to make a block. & eosin (H&E) reagent. Wet the stained tissue slide with mounting solution, attach a cover-slide, observe it under an optical microscope at ×100 magnification, and photograph it with a digital camera.

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

(4) inflammatory cytokine expression analysis

ELISA (Enzyme-Linked Immunosorbent Assay) was performed to measure the expression level of inflammatory cytokines (TNF-α, IL-1β) in bronchoalveolar lavage fluid (BALF). Specifically, put 50 µL of diluent assay buffer and 50 µL of BALF solution per well in a 96-well plate coated with Capture antibody, react for 2 hours, wash 4 times using wash buffer, and put biotin-conjugate antibody reagent in each well was treated at 100 μL and reacted at room temperature for 2 hours. After washing 4 times, each well was treated with substrate solution and darkly reacted 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.

As shown in Figure 5, it was confirmed that the significant reduction effect of TNF-α, one of the inflammatory cytokines acutely generated in the test animals administered in Examples 3 and 24. This is a result that supports the fact that the expression of inflammatory cytokines increases by increasing the expression of NF-kB in the respiratory tract by fine dust. Examples 3 to 24 in which the composition efficiently suppresses NF-kB expression and suggests that various inflammatory cytokines can be efficiently suppressed.

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

Lung tissue was extracted and rapidly frozen in liquid nitrogen and then stored frozen (-70 °C). For protein extraction in tissues, 0.5 mL of lysis buffer and 3 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°C for 1 hour, centrifugation was performed at 4°C, 16,000 × g for 15 minutes using a rotator to separate the protein between the fat layer and the pellet, and then 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 added to a 96-well plate, absorbance was measured at 595 nm wavelength, and then the BSA standard curve was used. The protein concentration was calculated.

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

ELISA (Enzyme-Linked Immunosorbent Assay) was performed to measure IL-6, an inflammatory cytokine in lung tissue. Specifically, put 50 µL of diluent assay buffer and 50 µL of BALF solution per well in a 96-well plate coated with Capture antibody, react for 2 hours, wash 4 times using wash buffer, and put biotin-conjugate antibody reagent in each well was treated at 100 μL and reacted at room temperature for 2 hours. After washing 4 times, each well was treated with substrate solution and darkly reacted 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 as shown in FIGS. 6 to 7 .

As shown in FIG. 6 , it was confirmed that the expression level of c-Caspase-3 protein, an apoptosis-related protein, decreased in the test group administered with Examples 3 and 24. In particular, it was involved in the endogenous mechanism of apoptosis and c-Caspase- The expression level of c-Caspase-9, an upper signaling protein of 3, also showed a tendency to decrease.

The expression pattern of IL-6, an inflammatory cytokine, in lung cells is shown in FIG. 7 . IL-6 is an inflammatory cytokine that is generated in the early stages of the inflammatory response and is known to promote 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 due to inflammation. Example 24 In the 100 mg/kg administration group, it was confirmed that the expression level of IL-6 in the lung cells stimulated by fine dust was significantly reduced, which prevented respiratory diseases and suggest that improvements can be made.

Formulation example

Formulation examples of the composition according to an aspect of the present invention will be described below, but other various formulations are also applicable, which is not intended to limit the present specification, but merely to describe it in detail.

[Formulation Example 1] Soft capsule

8 mg of the extract of Example, vitamin E 9 mg, vitamin C 9 mg, palm oil 2 mg, hydrogenated vegetable oil 8 mg, yellow wax 4 mg and lecithin 9 mg are 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. Separately, a soft capsule sheet was prepared in a ratio of 66 parts by weight of gelatin, 24 parts by weight of glycerin, and 10 parts by weight of sorbitol solution, and the filling solution was filled to prepare a soft capsule containing 400 mg of the composition according to the present specification.

[Formulation Example 2] Tablet

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 are mixed, granulated using a fluidized bed dryer, and 6 mg of sugar ester is added. 500 mg of these compositions are compressed in a conventional manner to prepare tablets.

[Formulation Example 3] Drink agent

After mixing 8 mg of the extract of Example, 9 mg of vitamin E, 9 mg of vitamin C, 10 g of glucose, 0.6 g of citric acid, and 25 g of liquid oligosaccharide, 300 ml of purified water is added, and 200 ml of each bottle is filled. After filling the bottle, it is sterilized 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, formed into granules using a fluidized bed granulator, and then filled in a bag to prepare granules.

[Formulation Example 5] Injection

Injections were prepared in a conventional manner according to the composition shown in Table 6 below.

compounding ingredients content Extracts of Examples 10-50 mg Sterile distilled water for injection appropriate amount pH adjuster appropriate amount

[Formulation Example 6] Health drink

Health drinks were prepared in a conventional manner according to the composition shown in Table 7 below.

compounding ingredients content Extracts of Examples 1000 mg citric acid 1000 mg oligosaccharide 100 g Taurine 1 g Purified water remaining amount

After mixing the above ingredients 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.

As mentioned above, although the present invention has been described through examples, experimental examples, and manufacturing examples, those of ordinary skill in the art to which the present invention pertains will realize that the present invention will be implemented 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)

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.
The method of claim 1,
The extract is water, an alcohol having 1 to 5 carbon atoms, or an aqueous solution of an alcohol having 1 to 5 carbon atoms as an extraction solvent, a pharmaceutical composition for preventing or treating fine dust stimulation respiratory disease.
3. The method of claim 2,
The extract is a water extract, a pharmaceutical composition for preventing or treating fine dust stimulation respiratory disease.
4. The method of claim 3,
The extract is extracted for 20 minutes to 10 hours at 50 ℃ to 150 ℃, a pharmaceutical composition for preventing or treating fine dust stimulatory respiratory disease.
The method of claim 1,
The citrus plants are pomelo (Citrus maxima), citrus (Citrus medica), mandarin (Citrus reticulate), tangja (Citrus trifoliate), gold persimmon (Citrus japonica), grapefruit (Citrus paradisi), citron (Citrus junos ) , lemon (Citrus) limon), lime (Citrus aurantifolia), tangerine (Citrus unshiu), wenzhou citrus (satsuma mandarin), and sweet orange (Citrus sinensis) any one or more of, a pharmaceutical composition for preventing or treating fine dust-stimulating respiratory diseases.
The method of claim 1,
The citrus genus plant is a fruit of a plant, a pharmaceutical composition for preventing or treating fine dust stimulation respiratory disease.
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.
8. The method of claim 7,
The extract is water, an alcohol having 1 to 5 carbon atoms or an aqueous solution of an alcohol having 1 to 5 carbon atoms as an extraction solvent, a food composition for preventing or improving fine dust stimulation respiratory disease.
9. The method of claim 8,
The extract is a water extract, a food composition for preventing or improving fine dust stimulation respiratory disease.
10. The method of claim 9,
The extract is extracted for 20 minutes to 10 hours at 50 ° C. to 150 ° C., a food composition for preventing or improving fine dust-stimulated respiratory diseases.
8. The method of claim 7,
The citrus plants are pomelo (Citrus maxima), citrus (Citrus medica), mandarin (Citrus reticulate), tangja (Citrus trifoliate), gold persimmon (Citrus japonica), grapefruit (Citrus paradisi), citron (Citrus junos ) , lemon (Citrus) limon), lime (Citrus aurantifolia), tangerine (Citrus unshiu), Wenzhou citrus (satsuma mandarin), and sweet orange (Citrus sinensis) any one or more, fine dust stimulation food composition for preventing or improving respiratory diseases.
8. The method of claim 7,
The citrus genus plant is a fruit of a plant, a food composition for preventing or improving fine dust-stimulated respiratory disease.

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