KR20200092532A - Anti-inflammatory composition using an extract of radish root, etc. - Google Patents

Abstract

Disclosed is an anti-inflammatory composition using an extract of radish root and the like. More specifically, the present invention proves that a mixture of a Raphanus sativus L. root extract, a whole Allium hookeri extract, an Acanthopanax sessiliflorum stem extract, a Dendropanax morbiferus leaf and stem extract, and/or a Codonopsis lanceolata root extract provides an obvious anti-inflammatory effect in an experiment using a mouse macrophage with an inflammatory reaction induced from lipopolysaccharide (LPS), which is ectoblast toxin, and an experiment using an animal model with an inflammatory reaction induced by using ovalbumin, LPS, and fine dust.

Description

Anti-inflammatory composition using an extract of radish root, etc.

The present invention relates to a composition for anti-inflammatory using radish root extract and the like.

Inflammation is a local biological defense response that appears in response to physical trauma, harmful chemicals, infections with bacteria, fungi, viruses, or pathological conditions caused by irritating substances in metabolites in vivo. Although various biochemical phenomena are involved in the inflammatory reaction, macrophage (Macrophage) produces oxidative nitric oxide (NO) and various inflammatory cytokines (TNF-α, IL-1β, IL-6) by chemical stimulation. It is known to play an important role in (Ito T., et al., Curr Drug Traget Inflamm Allergy, 2(3):257-265, 2003).

Non-steroidal anti-inflammatory drugs (NSAIDS), which are currently widely used as anti-inflammatory drugs, are known to cause serious side effects such as gastrointestinal disorders, liver disorders, and kidney disorders (Rainsford KD., Subcell biochem., 42:3) -27, 2007; Guruprasad P. Aithal., Rheumatology., 7:139-150, 2011; Praveen PN Rao et al., Pharmaceuticals., 3:1530-1549, 2010).

Therefore, it can be said that there is still a need for the development of new drugs with anti-inflammatory activity and low side effects and lasting effects.

The present invention discloses that radish root extract and the like have anti-inflammatory activity.

An object of the present invention is to provide a composition for anti-inflammatory using radish root extract and the like.

Other or specific objects of the present invention will be presented below.

The present inventors, as confirmed in the examples below, radish ( Raphanus sativus L.) root extract, three ( Allium hookeri ) outpost extract, agapi ( Acanthopanax sessiliflorum ) stem extract, the leaves and stems of the hwangchil tree ( Dendropanax morbiferus ) The mixture extract and/or the mixture of the root extract of Codonopsis lanceolata is tested with mouse macrophages in which the inflammatory response is induced by the bacterial outer membrane toxin LPS (lipopolysaccharide) and used with fine dust together with Ovalbumin and LPS In the experiment using an animal model inducing asthma and inflammatory reaction, it was confirmed that it has distinct anti-inflammatory activity and anti-asthmatic activity, and furthermore, these mixtures have anti-oxidative activity and GST, the second-phase detoxification enzyme of hepatocytes. (glutathione-S-transeferase) was confirmed to activate clearly.

In view of the above, the present invention, in one aspect, radish root extract, three kinds of outpost extract, stems of the extract of the stems, extract of the leaves and stem mixture of hwangchil tree, deodeok root extract and a mixture of two or more of these extracts as an active ingredient It can be grasped as a composition for anti-inflammatory, and in other aspects, radish root extract, three-sized outpost extract, stalk of stem extract, extract of leaves and stem mixture of hwangchil tree, deodeok root extract, and a mixture of two or more of these extracts as an active ingredient It can be grasped with the composition for antioxidant containing. In another aspect, as a composition for enhancing liver detoxification function, including radish root extract, three sage outpost extract, australian stem extract, hwangchil tree leaf and stem mixture extract, deodeok root extract and a mixture of two or more of these extracts as an active ingredient Can grasp.

In the anti-inflammatory composition of the present invention, the composition for antioxidant and the composition for enhancing liver detoxification function, the active ingredient is preferably a mixture of radish root extract, samchae outpost extract, stalk stem extract and extract of hwangchil tree leaf and stem mixture, In particular, when referring to the examples below, it is preferable that these mixing ratios are 30:30:10:30 by weight.

In addition, in the composition for antioxidant of the present invention, the active ingredient is preferably a mixture of radish root extract, samchae outpost extract, australian stem extract and deodeok root extract, and especially when referring to the examples below, the mixing ratio thereof is 50:20 It is preferable that it is: 10:20.

In the present specification, the term "extract" refers to water, lower alcohols having 1 to 4 carbon atoms (methanol, ethanol, butanol, etc.), methylene chloride, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol or a mixture obtained by leaching using a solvent, supercritical extraction solvent such as carbon dioxide or pentane It means the obtained extract or fraction obtained by fractionating the extract, and the extraction method can apply any method such as cold acupuncture, reflux, warming, ultrasonic radiation, supercritical extraction in consideration of the polarity, extraction degree, and storage degree of the active substance. have. In the case of fractionated extracts, the fractions obtained by suspending the extract in a specific solvent and mixing and policing with a solvent having a different polarity, adsorb the crude extract on a column filled with silica gel, etc., and then add a hydrophobic solvent, a hydrophilic solvent or a mixed solvent thereof. It means that it contains the fraction obtained as a mobile phase. In addition, the meaning of the extract includes a concentrated liquid extract or a solid extract in which the extraction solvent is removed by a method such as freeze drying, vacuum drying, hot air drying, spray drying, and the like.

In addition, in the present specification, "active ingredient" refers to a component that can exhibit a desired activity alone or itself can exhibit activity together with an inactive carrier.

Also, as used herein, "anti-inflammatory" is meant to include the improvement (reduction of symptoms), treatment, suppression or delay of the onset of such diseases as defined below.

In addition, in the present specification, the "inflammatory disease" refers to an inflammatory reaction characterized by a local or systemic bio-defense response to an external infection or autoimmunity such as external physical and chemical stimulation or infection of bacteria, fungi, viruses, and various allergens. It can be defined as the pathological symptom it causes. These inflammatory reactions include activation of various inflammatory mediators and enzymes associated with immune cells (eg iNOS, COX-2, etc.), secretion of inflammatory mediators (eg, secretion of NO, TNF-α, IL-6, etc.), body fluid infiltration, It involves a series of complex physiological reactions, such as cell migration and tissue destruction, and is manifested externally by symptoms such as erythema, pain, edema, fever, and a decrease or loss of certain functions in the body. The inflammatory disease may be acute, chronic, ulcerative, allergic or necrotic, so as long as any disease is included in the definition of such inflammatory disease, it is acute, chronic, ulcerative, allergic, or Necrotic or not. Specifically, the inflammatory diseases include asthma, allergic and non-allergic rhinitis, chronic and acute rhinitis, chronic and acute gastritis or enteritis, ulcerative gastritis, acute and chronic nephritis, acute and chronic hepatitis, chronic obstructive pulmonary disease, pulmonary fibroids , Irritable bowel syndrome, inflammatory pain, migraine, headache, back pain, fibromyalgia, fascia disease, viral infections (such as type C infection), bacterial infections, fungal infections, burns, wounds caused by surgical or dental surgery, pro Stargladin E hypersyndrome, atherosclerosis, gout, arthritis, rheumatoid arthritis, ankylosing spondylitis, Hodgkin's disease, pancreatitis, conjunctivitis, irisitis, scleritis, uveitis, dermatitis (including atopic dermatitis), eczema, multiple sclerosis, etc. Will be included.

Also, as used herein, "anti-asthma" is meant to include improvement (reduction of symptoms), treatment, suppression or delay in the development of such a disease.

The anti-inflammatory composition of the present invention, the composition for anti-oxidation, and the composition for enhancing liver detoxification function (hereinafter referred to as "the composition of the present invention") may exhibit improved activity of inflammatory diseases intended to be treated according to the use, formulation, etc. It may be included in any amount (effective amount) as long as it is, the typical effective amount will be determined within the range of 0.001% to 15% by weight based on the total weight of the composition. When the composition of the present invention is administered during the administration period according to the recommendation of a medical expert or the like, the "effective amount" refers to a mammal, preferably a human subject, the treatment of which is effective, or the suppression of the development of such pathological symptoms ·It refers to the amount of active ingredients that can have the intended medical and pharmacological effects such as delay. Such effective amount can be determined empirically within the range of ordinary skill in the art.

In the specific aspect, the composition of the present invention can be grasped as a food composition.

The food composition of the present invention can be produced in any form, such as tea, juice, carbonated beverages, beverages such as ionic beverages, processed oils such as milk, yogurt, gums, rice cakes, sweets, bread, cookies, noodles, etc. Food, tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, bars, etc. can be prepared as health functional food formulations.

In addition, the food composition of the present invention can be classified into any product as long as it complies with the enforcement regulations at the time of manufacture and distribution in terms of legal and functional classification. For example, it is a health functional food pursuant to the Korean Act on Health Functional Food, or a confectionary, soybean, tea, or beverage according to each food type in the Food Fair of the Korean Food Sanitation Act (「Food Standards and Standards」) , Special-purpose food.

The food composition of the present invention may include a food additive in addition to the active ingredient. Food additives can be generally understood as substances added to foods to be mixed or infiltrated in manufacturing, processing, or preserving foods, and their safety must be ensured because they are consumed daily and for a long time with foods. Food additives in accordance with national laws governing the manufacture and distribution of food ("Food Sanitation Act" in Korea) are limited in terms of ingredients or functions in terms of food additives with guaranteed safety. In the Korean Food Additives Fair (「Food Additive Standards and Standards」 published by the Ministry of Food and Drug Safety), food additives are classified into chemical synthetic products, natural additives, and mixed preparations in terms of ingredients, and these food additives are sweeteners and flavors in terms of functionality. It is divided into agents, preservatives, emulsifiers, acidulants, and thickeners.

Sweeteners are used to impart moderate sweetness to food, and both natural and synthetic can be used in the composition of the present invention. Preferably, a natural sweetener is used. Examples of the natural sweetener include sugar syrup such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

Flavoring agents can be used to enhance taste or aroma, and both natural and synthetic ones can be used. Preferably, it is the case of using a natural thing. In addition to flavor, when using natural ones, the purpose of enhancing nutrition can also be combined. As a natural flavoring agent, it may be obtained from apples, lemons, citrus fruits, grapes, strawberries, peaches, or the like, or may be obtained from green tea leaves, perilla, large leaves, cinnamon, chrysanthemum leaves, jasmine, and the like. In addition, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, and ginkgo can be used. Natural flavors may be liquid concentrates or solid extracts. In some cases, synthetic flavoring agents may be used, and synthetic flavoring agents may include esters, alcohols, aldehydes, terpenes, and the like.

Sodium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc. can be used as a preservative, and acacia gum, carboxymethylcellulose, xanthan gum, etc. Pectin and the like, and as the acidulant, arithmetic, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid and the like can be used. The acidulant may be added so that the food composition has an appropriate acidity for the purpose of suppressing the growth of microorganisms in addition to the purpose of enhancing taste.

As a thickener, a suspending agent, sedimentation agent, gel forming agent, swelling agent, etc. may be used.

The food composition of the present invention may include, in addition to the food additives as described above, physiologically active substances or minerals known in the art for the purpose of supplementing and reinforcing functional and nutritional properties, and having stability as food additives.

Examples of such bioactive substances include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, dibenzoyl thiamine, and the like, and calcium preparations such as calcium citrate and magnesium stearate as minerals Magnesium preparations such as iron, iron preparations such as iron citrate, chromium chloride, potassium iodide, selenium, germanium, vanadium, zinc, and the like.

The food composition of the present invention may include the food additives as described above in an appropriate amount to achieve the purpose of addition according to the product type.

With regard to other food additives that may be included in the food composition of the present invention, it is possible to refer to national foods or food additives.

The composition of the present invention may be identified as a pharmaceutical composition in other specific embodiments.

The pharmaceutical composition of the present invention may be prepared in an oral dosage form or a parenteral dosage form according to the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. Here, the route of administration may be any suitable route including a local route, an oral route, an intravenous route, an intramuscular route, and direct absorption through mucosal tissue, and two or more routes may be used in combination. An example of a combination of two or more routes is when two or more formulations of the drug are combined according to the route of administration, for example, when one drug is administered by the intravenous route and the second drug is administered by the local route.

Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or formulation, and specifically refer to the pharmacopeia of each country, including "Korea Pharmacopoeia".

When the pharmaceutical composition of the present invention is prepared in an oral dosage form, powders, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, suspensions, wafers according to methods known in the art with suitable carriers And the like. At this time, examples of suitable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, xylitol, starches such as corn starch, potato starch, wheat starch, cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, Celluloses such as hydroxypropyl methylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, ethanol, green Serol, etc. are mentioned. In the case of formulation, if necessary, suitable binders, lubricants, disintegrants, colorants, diluents, etc. may be included. Suitable binders include starch, magnesium aluminum silicate, starch ferist, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweetener, sodium alginate, polyethylene glycol, wax, etc., and lubricants include oleic acid Sodium, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, magnesium salts and calcium salts thereof, polydetylene glycol, and the like, and starch and methyl cellulose as disintegrants , Agar, bentonite, xanthan gum, starch, alginic acid or its sodium salt. Moreover, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine etc. are mentioned as a diluent.

When the pharmaceutical composition of the present invention is prepared in a parenteral dosage form, it may be formulated in the form of injections, transdermal administrations, nasal inhalants and suppositories according to methods known in the art with suitable carriers. When formulated as an injectable agent, an aqueous isotonic solution or suspension may be used as a suitable carrier. Specifically, an isotonic solution such as PBS (phosphate buffered saline) containing triethanol amine, sterile water for injection, or 5% dextrose may be used. . When formulated as a transdermal dosage form, it can be formulated in the form of ointments, creams, lotions, gels, external solutions, pasta, linen agents, aerosols, and the like. For nasal inhalants, dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc. can be formulated in the form of an aerosol spray using a suitable propellant. witepsol), tween 61, polyethylene glycols, cacao butter, laurin, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate, sorbitan fatty acid esters, and the like.

Regarding the specific formulation of the pharmaceutical composition, it is known in the art, and for example, see Remington's Pharmaceutical Sciences (19th ed., 1995) and the like. The above documents are regarded as part of this specification.

Preferred dosages of the pharmaceutical compositions of the present invention range from 0.001 mg/kg to 10 g/kg per day, preferably 0.001 mg/kg to 1 g per day, depending on the patient's condition, weight, sex, age, patient severity, and route of administration. /kg range. Administration can be made once a day or divided into several times. Such dosage should not be construed as limiting the scope of the invention in any aspect.

The composition of the present invention, in particular, the anti-inflammatory composition and the antioxidant composition, in another specific embodiment, can be identified as a cosmetic composition. When the anti-inflammatory composition of the present invention is identified as a cosmetic composition, its use may be understood as a use for suppressing inflammatory skin trouble, alleviating inflammatory skin irritation, and the like.

Even if the composition of the present invention is identified as a cosmetic composition, the cosmetic composition can be classified into any product in accordance with its use, and in particular, functional cosmetics having non-functionality such as improvement of skin trouble, atopic dermatitis, etc. It may be a general cosmetic. In the form of a product, any type of product can be taken. Specifically, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, and waxes It can take the form of products such as foundations and sprays. In a specific product form, it may be flexible lotion, nutrition lotion, nutrition cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder formulation.

The cosmetic composition of the present invention may include, in addition to its active ingredients, ingredients commonly used in cosmetic compositions, such as stabilizers, solubilizers, surfactants, vitamins, pigments and conventional auxiliary agents such as pigments, and carriers.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, trakant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide, etc. may be used as a carrier component. Can.

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additionally chlorofluorohydrocarbon, propane /Propellant such as butane or dimethyl ether.

When the formulation of the present invention is a solution or an emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, specifically water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, fatty acid ester of sorbitan, and the like can be used.

When the formulation of the present invention is a suspension, liquid diluents such as water, ethanol or propylene glycol as carrier components, ethoxylated isostearyl alcohol, suspending agents such as polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, microcrystals Sex cellulose, aluminum metahydroxide, bentonite, agar and the like can be used.

When the formulation of the present invention is a surfactant-containing cleansing, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivatives, methyltaurate, sarcosinate, fatty acid amide as a carrier component Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

The cosmetic composition of the present invention can be prepared according to the manufacturing method of the cosmetic composition that is conventionally performed in the art, except that it contains the active ingredient.

As described above, according to the present invention, it is possible to provide an anti-inflammatory composition using radish root extract or the like.

The anti-inflammatory composition of the present invention can be commercialized as food, cosmetics, drugs, etc., for the purpose of improving inflammatory diseases and for alleviating inflammatory skin irritation.

1 is a result of examining cytotoxicity in mouse-derived macrophages RAW264.7.
2 and 3 are the results of measuring the production of inflammatory factors (NO and TNF-α) in mouse-derived macrophages RAW264.7.
4 and 5 are the results of measuring the degree of production of inflammatory cytokines and the like in an inflammatory animal model experiment.
6 and 7 are the results of the antioxidant activity experiment.
8 is a result of GST activity enhancement experiments.

Hereinafter, the present invention will be described with reference to Examples. However, the scope of the present invention is not limited to these examples.

<Example> Anti-inflammatory activity experiment, antioxidant activity experiment, liver detoxification function enhancement experiment

1. Materials and Methods

1.1 Materials

The extraction targets were radish ( Raphanus sativus L.) roots, allium hookeri outposts, acanthopanax sessiliflorum stems, leaf and stem mixtures of Dendropanax morbiferus , and Codonopsis lanceolata roots.

For the extract, 10 L of water was added to 1 kg of each extraction object prepared as a powder, and hot water was extracted using a reflux extractor at 100° C. for 5 hours, and the hot water extract was filtered with Whatman No. 2, then concentrated under reduced pressure and frozen. It was dried to obtain a powder.

Each extract thus obtained was mixed in a weight ratio as shown in the table below and used in the experiment.

1.1 Anti-inflammatory active cell experiment

(1) Cell culture

RAW264.7 cells, which are mouse-derived macrophages, were cultured in DMEM (Dulbecco's modified Eagle's medium) medium containing 10% FBS and 1% penicillin-streptomycin. Cultured at 37°C in the presence of 5% carbon dioxide.

(2) Cytotoxicity evaluation

After RAW 264.7 cells were cultured at a concentration of 1×10 ⁵ cells/well for 24 hours, samples of various concentrations were treated. After 2 hours, cytotoxicity was measured after stimulation at a concentration of lipopolysaccharide (LPS, 1 mg/mL) for 24 hours. For cytotoxicity, CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) diluted 10-fold in medium was dispensed for 100 mL, and reacted for 1 hour to measure absorbance at 490 nm.

(3) Measurement of NO production

NO nitrogen in the culture was measured using Griess analysis. The culture supernatant and the same amount of Griess reagent (a mixture of 5% phosphoric acid and 1% salfanilamide) were added, reacted at room temperature for 20 minutes, and absorbance was measured at 540 nm.

(4) Measurement of cytokine production

RAW 264.7 cells were dispensed at a concentration of 2.5×10 ⁵ cells/well in 24 wells, and cultured for 24 hours. After removal of the culture supernatant, samples of various concentrations were processed. After 2 hours, stimulation was performed with LPS for 24 hours, and the amount of TNF (Tumor necrosis factor)-a in the culture supernatant was measured by an Enzyme-linked immunosorbent assay (ELISA) method.

1.2 Anti-inflammatory animal test

(1) Experimental animals

Animal experiments were conducted in the animal breeding room of Dongshin University College of Oriental Medicine (humidity 40-60%, temperature 24-26 ℃) by dividing the weight of 160-190 g of Sprague Dawley-based white paper mice by weight. The feed was free to eat solid feed (Pellet, GMO, Korea) and supplied enough water. The group was separated again after restoring for more than 4 days.

(2) Test pretreatment process

Experimental mouse increased irritable respiratory-related asthma and inflammatory response For induction, Ovalbumin (OVA, 1 mg/kg) + Lipopolysaccharide (LP, 5 mg/kg) was diluted with physiological saline for injection, and then administered intraperitoneally twice every 5 days to sensitize the whole body. At this time, OVA was used after dissolving 10 mg/kg aluminum potassium sulfate (Sigma-Aldrich Co.) in phosphate buffer (PBS). After intraperitoneal administration, they were allowed to rest for 5 days (Jang et al., 2015).

In addition, the exposure test of fine dust, an air pollutant, dissolved 2% fine dust (Standard Reperance Matter, NIST, USA) + 3% ovalbumin + 5% Lipopolysaccharide in physiological saline on the 6th day after intraperitoneal administration. Tech, Korea) for a total of 4 times and 60 minutes a day (total doses of 15 minutes once every 4 hours). Then, after intraperitoneal administration, samples were treated by misting a total of 4 times on the 7th, 9th, and 11th.

(3) Test treatment tool

The experimental treatment did not process anything (normal), fine dust+OVA+LPS(control), fine dust+OVA+LPS+dexamethasone (DEXA, positive control, 3 mg/kg), fine dust+OVA+LPS+ sample 100 mg/kg (HS100), fine dust+OVA+LPS sample 200 mg/kg (HS200), fine dust+OVA+LPS sample 400 mg/kg (HS400) A total of 6 treatment groups were used, and the number of repetitions was treated with 5 animals (n=5). Here, SSA4 was used as the sample.

(4) Cytokine change analysis-ELISA

Expression levels at the protein level of inflammatory cytokines (TNF-α, IL-1β, IL-6, and IFN-γ) were confirmed. Blood collected from mice was measured using serum centrifuged at 3,000 rpm for 30 min in a centrifuge (Mega21, Hanil, Korea), and a sandwich ELISA mouse kit (R&D system, Mckinley place NE, USA) was used.

(5) Cytokine change analysis: RT-PCR

In order to measure changes in substances related to inflammation caused by air pollution, total RNA was extracted from the lung tissue using an extraction kit, and changes in mRNA were observed through a PCR method. 5 μg of isolated total RNA, 2.5 μl Oligo (dT), and DEPC-treated water were added to RT premix (Bioneer, Korea) to synthesize 50 μl cDNA using Mastercycler gradient (Eppendorf, Germany), and using the primers below. PCR (GeneAmp PCR system 9700, Applied Biosystems, USA) was performed.

IL-1β: (F) 5'-ATGGCAACTGTTCCTGAACTCAACT-3', (R) 5'-CAGGACAGGTATAGATTCTTTCCTTT-3'

IL-6: (F) 5'-TTGCCTTCTTGGGACTGATG-3' (F), (R) 5'-CAGAATTGCCATTGCACAACT-3'

IFN-γ: (F) 5'-AATGAACGCTACACACTGCA-3', (R) 5'-TGAAGAAGGTAGTAATCAGG-3'

TNF-α: (F) 5'-CCACATCTCCCTCCAGAAAA-3', (R) 5'-AGGGTCTGGGCCATAGAACT-3'

1.3 Antioxidant activity experiment

(1) DPPH radical scavenging ability

Samples of various concentrations are dissolved in a methanol solvent, and mixed with 900 μL of DPPH solution (100 μM) and 100 μL of each sample and stirred. After reacting the mixed sample in the dark for 30 minutes, the absorbance was measured at 517 nm. DPPH radical scavenging activity was calculated through a 3 replicate experiment as a percentage of the sample-untreated group. As a standard sample, gillic acid was used.

(2) ABTS cation radical scavenging ability measurement

7 mM ABTS [2,2-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) diammonium salt] solution and 2.45 mM potassium persulfate were mixed at a final concentration and allowed to stand at room temperature for one day, and then ABTS radical generation was induced. . For the ABTs radical, PBS (phosphate buffer saline, pH 7.4) buffer was used as a diluent to make the absorbance value at 732 nm to be 0.7 nm. 200 μL of ABTS solution diluted in a 96 well plate and 100 μL of sample (dissolved in MeOH) were diluted by concentration, placed in a microplate reader after 1 minute, and absorbance was measured at 732 nm. The ABTS radical scavenging activity was calculated as a percentage of the sample-untreated group through 3 replicates. As a standard sample, gillic acid was used.

1.4 Liver detoxification activity (GST) experiment

(1) Cell culture

As the cell line used in this experiment, hepa1c1c7 (murine hepatoma cell line) cells derived from a passage-preserved mouse were used with MEM medium containing 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin/streptomycin). Incubated 2-3 times a week in a 37"C incubator with 5% CO ₂ .

(2) Intracellular GST activity

Intracellular GST activity was measured by measuring the number of cells with 0.4% trypan blue staining for 1 x 10 ⁴ cells. After dispensing 200 μL into 96-well microtiter plates at a concentration of cells/well, the cells were cultured for 24 hr. The medium was removed, and 200 μL was added to the new MEM medium and samples were added, followed by incubation for 48 hours. The medium was removed again and washed 4 times with PBS to lyse the cells. The lysed cells were added to 50% of 2% triton X-100 solution and mixed for 10 minutes. GST was added with 100 μL of 2.5 mM GSH and l mM CDNB (1-chloro-2, 4-dinitrobenzene) reaction solution dissolved in 0.1 M potassium phosphate buffer, mixed for 1 minute, and absorbance was measured at 405 nm for 5 minutes. The GST activity was calculated as slop/min/mg protein and expressed as the ratio of the GST activity of the sample to the GST activity of the control group, which is a sample-free group.

Statistics processing

All measured values were expressed as mean and standard error using the Excel statistic program (Microsoft, USA). Statistical analysis between each experimental group was non-parametric using SPSS for Windows (SPSS, USA). The Mann-Whitney U test was performed. Each experimental group was tested for significance at α=0.05 level (P<0.05) and α=0.01 level (P<0.01) compared to the control group.

2. Experimental results

2.1 Anti-inflammatory cell experiment

The results of the cytotoxicity experiment are shown in FIG. 1, and the degree of NO production and TNF-α production are shown in FIGS. 2 and 3. All the mixture samples did not show cytotoxicity and inhibited NO production and TNF-α production in a concentration-dependent manner. In particular, the D(SSA4) sample was highly active (A: SSA1, B: SSA2, C:SSA3, D:SSA4). In animal experiments, only the D sample was used for this reason.

2.2 Anti-inflammatory active animal experiment

The ELISA measurement results are shown in Fig. 4, and the RT-PCR measurement results are shown in Fig. 5. In both the ELISA measurement result and the RT-PCR measurement result, the samples (SSA4, HS) inhibited the production of inflammatory cytokines and the like in proportion to the dose.

2.3 Antioxidant activity

DPPH radical scavenging activity and ABTS radical scavenging activity are shown in FIGS. 6 and 7. 6 and 7, it can be seen that the SSA1 sample has the highest activity.

2.2 Liver detoxification activity test

The results of the liver detoxification enhancing activity experiment are shown in FIG. 8. In liver detoxification enhancing activity, SSA4 showed the highest activity.

Claims (8)

An anti-inflammatory food composition comprising radish root extract, samchae outpost extract, agapi stem extract, extract of hwangchil tree leaf and stem mixture, deodeok root extract and a mixture of two or more of these extracts as an active ingredient.
According to claim 1,
The mixture is characterized in that the mixture of the radish root extract, the extract of the three plants, the extract of the stem of the stem and the extract of the leaves and stem mixture of hwangchil tree is a mixture in a weight ratio of 30:30:10:30.
According to claim 1,
The extract is a composition characterized in that the extract of water, ethanol or a mixed solvent thereof.
An anti-inflammatory food composition comprising radish root extract, samchae outpost extract, agapi stem extract, extract of hwangchil tree leaf and stem mixture, deodeok root extract and a mixture of two or more of these extracts as an active ingredient.
According to claim 4,
The mixture is characterized in that the mixture of the radish root extract, the extract of the three plants, the extract of the stem of the stem and the extract of the leaves and stem mixture of hwangchil tree is a mixture in a weight ratio of 30:30:10:30.
According to claim 4,
The extract is a composition characterized in that the extract of water, ethanol or a mixed solvent thereof.
A mixture of radish root extract, samchae outpost extract, agapi stem extract and deodeok root extract, but as an active ingredient, the mixing ratio thereof is 50:20:10:20, characterized in that the antioxidant composition.
A composition for enhancing liver detoxification function, comprising radish root extract, samchae outpost extract, australian stem extract, and a leaf and stem mixture of hwangchil tree as an active ingredient, and the mixing ratio is 30:30:10:30 by weight.

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