KR102180527B1 - A composition for antioxidating and antiinflammation comprising extracts of red ginseng and aronia melanocarpa - Google Patents

Abstract

The present invention relates to a composition comprising a red ginseng extract and an aronia extract as an active ingredient, and the composition of the present invention comprising a red ginseng extract and an aronia extract as an active ingredient has a high content of total polyphenols, flavonoids and condensed tannins, , Radical scavenging ability and electron donating ability are excellent, so it has an antioxidant effect, and it has an anti-inflammatory effect by inhibiting the production of NO and inflammatory cytokines. In addition, the red ginseng extract and aronia extract of the present invention do not have cytotoxicity and skin side effects, so they can be safely used in cosmetics, pharmaceutical and food compositions.

Description

Antioxidant and anti-inflammatory composition comprising red ginseng and aronia extract {A COMPOSITION FOR ANTIOXIDATING AND ANTIINFLAMMATION COMPRISING EXTRACTS OF RED GINSENG AND ARONIA MELANOCARPA}

The present invention relates to a composition comprising a red ginseng extract and an aronia extract as an active ingredient, and more specifically, an antioxidant and anti-inflammatory pharmaceutical composition, a food composition and a cosmetic composition comprising red ginseng extract and aronia extract as an active ingredient It is about.

Recently, as the level of living and income has improved, a lot of interest has been increasing to improve the quality of life. In particular, interest in the development of physiologically active substances for metabolism and internal diseases including antioxidants has increased as a countermeasure against aging, various inflammatory diseases and cancer (KB Kim, et al., Journal of Nutrition and Health. 50(5)) ;415-425 (2017)). Biochemical oxidation reactions are constantly occurring to supply the necessary energy in the living body, and reactive oxygen species (ROS), which is called harmful oxygen, generated during this process is the most stable form of oxygen, triplet oxygen, during oxidation and reduction. Unstable free radicals such as superoxide anion, hydroxyl radical, hydrogen peroxide, and hydrogen peroxide (H ₂ O ₂ ) are unstable and oxidizing power Because it is highly highly reactive with biological substances, if it cannot be removed from the human body, it causes oxidative stress. This oxidative stress is linked to the inflammatory reaction, thus inducing lipid peroxidation in various metabolic processes, protein, and By inducing aging and transformation of cells by damaging cell membranes and DNA, it causes various diseases such as stroke, cancer, arteriosclerosis, Alzheimer's disease, Parkinson's disease, and arteriosclerosis (Valko M, et al., Int J Biochem Cell Biol. 39:44-84 (2007); And Halliwell B, et al., NY, USA. 968 (1999)) As a physiological antioxidant enzyme that protects the living body by removing the free radicals thus generated, superoxide dismutase ( superoxide dismutase (SOD), catalase, glutathione-peroxidase (GSHpx), glutathione S-transferase (GST), etc., and small molecule antioxidants or free radical scavengers. Α-tocopherol, β-carotene, ascorbic acid, glutathione, etc. are known to do so (Kim SM, et al., Life Sci. 90(21-22);874-882(2012)).

Therefore, in recent years, by removing such ROS, the use of antioxidants to protect the oxides generated by oxidative stress in the living body is increasing, and in particular, the importance of natural antioxidants widely distributed in natural products and research on this increase. Are doing.

On the other hand, the inflammatory reaction is a biodefensive reaction against external physical or chemical stimuli or bacterial infection. Nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL), called inflammatory mediators. Produces and secretes -1β, granulocyte-macrophage colony-stimulating factor (GM-CSF), and various inflammatory cytokines such as IL-6 (YC Yoo, et al., Journal of Life Science. 26(3);338-345(2016)). In addition, by activating nuclear factor kappa B (NF-κB), an inflammatory transcription factor, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are activated. ), prostaglandin E2 (PGE2) is produced, and the secreted inflammatory cytokine activates NF-κB again to amplify the cytokine cascade to expand the inflammatory state. In particular, NO may exhibit antibacterial and antitumor effects, but if excessively produced and secreted due to pathological causes, it may lead to a risk of septic shock and nerve tissue damage due to excessive vasodilation (S Han, et al., Immunopharmacol. Immunotoxicol. 35;34-42 (2013)). These inflammatory cytokines and inflammatory mediators play an important role in the inflammatory process, and appropriately controlling their abnormal production has been proposed as a treatment for inflammatory diseases.

Red ginseng (Red ginseng) belongs to the ginseng genus of the family Ogalpiaceae and is evaluated as the world's best health food or medicine due to its various pharmacological actions. It is mainly composed of various components such as saponin, essential oil, polyacetylene, phenol, alkaloids, polysaccharides, amino acids, organic acids, and vitamins, and is already widely known for enhancing liver function, antidiabetic effect, improving cardiovascular system, and antioxidant anti-inflammatory effects (DS Kim, et al., J Ginseng Res. 30(3);117-127(2006)).

On the other hand, Aronia (Aronia melanocarpa) is a plant fruit of berries belonging to the Rosaceae family, which grows wild in North America, and is also called black chokeberry. Aronia berry contains a large amount of anthocyanin and other flavonoids, polyphenol oil, and tannin, and is recently known to have antioxidant, anti-inflammatory, anti-diabetic, immunomodulatory, and various physiological activities (HM Lee , et al., J. Soc. Cosmet. Scientists Korea. 39(4);337-345 (2013)).

Accordingly, the present inventors studied the efficacy of the mixture of red ginseng and aronia more closely in the antioxidant and anti-inflammatory activity, and found that the mixed extract of red ginseng and aronia had very excellent antioxidant and anti-inflammatory activity. Was completed.

Accordingly, a technical problem to be solved in the present invention is to provide a material having anti-oxidation and anti-inflammatory effects and excellent human stability.

In order to solve the above technical problem, the present invention provides an antioxidant and anti-inflammatory composition comprising red ginseng extract and aronia extract.

Preferably, the composition comprising the red ginseng extract and the aronia extract in the present invention may be a pharmaceutical composition, a food composition or a cosmetic composition.

Preferably, in the present invention, the red ginseng extract and the aronia extract may be mixed in a weight ratio of 1:1 to 4:1, more preferably in a weight ratio of 2:1.

The term "extract" of the present invention refers to a preparation obtained by squeezing a herbal medicine into an appropriate leachate and evaporating the leachate, but is not limited thereto, but is not limited to the extract obtained by the extraction treatment, the diluted solution or the concentrate of the extract, or the extract. It may be a dried product obtained by drying, a preparation thereof, or a purified product.

The red ginseng extract and aronia extract of the present invention can be prepared using a general extraction method, separation and purification method known in the art. As the extraction method, a method such as hot water extraction, hot water extraction, cold needle extraction, reflux cooling extraction, or ultrasonic extraction may be used, but is not limited thereto.

According to a specific embodiment of the present invention, the red ginseng extract and the aronia extract are composed of an extraction solvent such as water, an anhydrous or aqueous lower alcohol having 1 to 3 carbon atoms, and a mixed solvent thereof after pulverizing the obtained red ginseng and aronia, respectively. It can be extracted using a solvent selected from the group as an extraction solvent, and the amount of the extraction solvent may be 2 to 20 times by weight, preferably 5 to 15 times by weight of the dry weight of the herbal medicine.

According to a specific embodiment of the present invention, the red ginseng extract is washed with water, and then water, ethanol or a mixture thereof, preferably alcohol (ethanol) is used at 60 to 80°C, preferably 70 to 75°C. It can be obtained by extraction for 5 to 20 hours, preferably 10 to 15 hours, and then filtration and concentration.

According to one embodiment of the present invention, the aronia extract may be aronia concentrate or aronia vinegar.

The aronia concentrate may be prepared by juicing the aronia fruit, filtration and aging at low temperature, mixing with purified water, aging at low temperature, filtration and concentration.

The aronia vinegar is a product prepared by mixing the aronia concentrate and the pear concentrate, followed by alcohol fermentation using a yeast strain, and then acetic acid fermentation using an acetic acid strain.

According to a specific embodiment of the present invention, the pear concentrate may be prepared by mixing and extracting pears and purified water, followed by filtration and concentration.

The composition of the present invention may contain 0.1 to 90% by weight, more preferably 10 to 80% by weight, based on the total weight of the red ginseng extract and the aronia extract. At this time, if the content of red ginseng extract and aronia extract is less than 0.1% by weight, the antioxidant and anti-inflammatory effects, which are the target effects of the present invention, cannot be obtained, and if it exceeds 90% by weight, the effect is not proportional with the increase in the content. There is a problem that it may be inefficient and the stability of the formulation is not secured.

The composition containing the red ginseng extract and the aronia extract of the present invention exhibits antioxidant and anti-inflammatory effects, and as a natural substance, has little cytotoxicity.

According to one embodiment of the present invention, it provides a pharmaceutical composition comprising red ginseng extract and aronia extract as active ingredients.

The pharmaceutical composition according to the present invention includes a pharmaceutically acceptable carrier in addition to red ginseng extract and aronia extract. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used at the time of formulation, and are lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. It does not become. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, etc. by various routes such as oral or parenteral, such as oral, rectal or intravenous, muscle, subcutaneous, intrauterine dural or cerebrovascular Can be administered by intra-injection. Preferably, it can be administered by oral administration by feeding.

A suitable dosage of the pharmaceutical composition of the present invention is variously prescribed depending on factors such as formulation method, mode of administration, age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and response sensitivity. Can be. The dosage of the pharmaceutical composition of the present invention is in the range of 0.001-100 mg/kg on an adult basis. In addition, in the case of an external preparation, it is recommended to apply once to 5 times a day in an amount of 1.0 to 3.0 ml based on an adult and continue for more than 1 month. However, the dosage is not intended to limit the scope of the present invention.

The pharmaceutical composition of the present invention is prepared in unit dosage form or in a multi-dose form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person skilled in the art. It can be manufactured by incorporating it into a container. In this case, the formulation may be in the form of a solution, suspension, syrup, or emulsion in an oil or aqueous medium, or may be in the form of an excir, powder, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.

According to one embodiment of the present invention, it provides a food composition comprising red ginseng extract and aronia extract as active ingredients.

The food composition according to the present invention may further include red ginseng extract and aronia extract as active ingredients, as well as ingredients commonly added during food production, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents. I can.

Examples of the carbohydrate include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents [taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used.

For example, when the food composition of the present invention is prepared as a drink, in addition to the red ginseng extract and aronia extract of the present invention, a pear concentrate, a mixed plant extract, for example, astragalus, cinnamon, angelica, licorice, baekchul, donggulle, reishi mushroom extract, Polydextrose, vitamin C, nicotinic acid amide, trisodium citrate, anhydrous citric acid, and the like may be further included.

According to one embodiment of the present invention, it provides a cosmetic composition comprising red ginseng extract and aronia extract as active ingredients.

In the cosmetic composition of the present invention, in addition to the red ginseng extract and aronia extract as active ingredients, ingredients commonly added to the cosmetic composition, such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and flavors, and carriers Can be added as.

The cosmetic composition of the present invention may be prepared in any formulation commonly prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing , Oil, powder foundation, emulsion foundation, wax foundation, spray, etc. may be formulated, but is not limited thereto. In more detail, it may be prepared in the form of a nourishing cream, astringent lotion, a soft lotion, lotion, essence, a nourishing gel or a massage cream.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth gum, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide are used as carrier components. Can be.

When the formulation of the present invention is a powder or a spray, toss, 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 / May contain propellants such as butane or dimethyl ether.

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

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 ester and polyoxyethylene sorbitan ester, crystallites Sex cellulose, aluminum metahydroxide, bentonite, agar or tracanth gum, and the like can be used.

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

The cosmetic composition of the present invention further comprises fatty substances, organic solvents, solubilizing agents, thickening and gelling agents, softening agents, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, film forming agents, water, Ionic or nonionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives, lipid vesicles or any commonly used in cosmetics It may contain adjuvants commonly used in the field of cosmetology or dermatology, such as other ingredients of. And, the above components may be introduced in an amount generally used in the field of dermatology.

On the other hand, the red ginseng extract and aronia extract of the present invention are natural substances that are harmless to the human body, and have little toxicity and side effects, so they can be safely used even when used for a long period of time, and are particularly safely applied to cosmetic, pharmaceutical and food compositions as described above. can do.

As described above, the composition comprising the red ginseng extract and aronia extract of the present invention as active ingredients has a high content of total polyphenols, flavonoids, and condensed tannins, has excellent radical scavenging ability and electron donating ability, has an antioxidant effect, and produces NO and It has an anti-inflammatory effect by inhibiting the production of inflammatory cytokines. In addition, the red ginseng extract and aronia extract of the present invention do not have cytotoxicity and skin side effects, so they can be safely used in cosmetics, pharmaceutical and food compositions.

1 shows the measurement results of electron donating ability of red ginseng extract and aronia extract.
2 is a result of measuring the superoxide dismutase (SOD)-like activity of red ginseng extract and aronia extract.
Figure 3 shows the measurement results of cation radical scavenging ability of red ginseng extract and aronia extract.
Figure 4 shows the measurement results of the superoxide anion radical scavenging ability of red ginseng extract and aronia extract.
Figure 5 shows the measurement results of the FRAP analysis of red ginseng extract and aronia extract.
6 shows the measurement results of reducing power of red ginseng extract and aronia extract.
7 shows the cytotoxicity of red ginseng extract and aronia extract.
Figure 8 shows the NO inhibitory activity of red ginseng extract and aronia extract.
Figure 9 shows the effect of red ginseng extract and aronia extract on inflammatory cytokines (Cytokine).

Hereinafter, examples, etc. will be described in detail to aid understanding of the present invention. However, the embodiments according to the present invention may be modified in various other forms, and the scope of the present invention should not be construed as being limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

Materials and reagents

DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diamonium salt), Iron (Ⅲ) chloride, Potassium ferricyanide, Trichloroacetic acid, Iron( Ⅱ) chloride, 2,2'-Bipyridyl, Copper (Ⅱ) chloride, Ammonium acetate, Pyrogallol, Quercetin, Folin & Ciocalteu's phenol reagent, Gallic acid, Vanillin, Catechin, TPTZ (2,4,6-tripyridyl-S-triazine ), Ferric Chloride, Xanthine oxidase, and Nitroblue tetrazolium were purchased and used from Sigma-Aldrich (St. Louis, Mo, USA), respectively. Glacial acetic acid and Xanthine were purchased from Alfa Aesar and used, and sodium acetate trihydrate was purchased from JUNSEI. RPMI Medium 1640, penicillin-streptomycin, phosphate buffered saline (PBS), and fetal bovine serum (FBS) used for cell culture were purchased from Hyclone, and Griess Reagent kir was purchased from Promega Corporation. CCK-8 (Cell Counting Kit-8) was purchased and used from Dojindo. For the solvents and reagents used in other studies, first-class and special-grade reagents were purchased and used.

<Example 1> Preparation of a composition containing red ginseng extract and aronia extract

(1) Preparation of red ginseng extract

As a raw material for red ginseng, red ginseng of 4 years old or more suitable for standards and standards set by the health functional food process and food code was checked, and then selected and purchased. After selecting foreign substances from the purchased red ginseng, they were put into an extractor and washed with water. After adding 7-8 times the amount of alcohol (95%) of the washed red ginseng raw material, it was extracted over 3-4 times at 70-75°C for 12 hours or more. The obtained red ginseng extract was filtered through a centrifugal filter of 12,000 rpm or higher, and then the concentrated solution was stirred and sterilized at 70±2° C. for 4 hours or longer in a sterilization device, and then filled and sealed according to packaging specifications.

(2) Preparation of aronia vinegar

Aronia fruit was used as a raw material to sort foreign substances, washed with water, and then the aronia peel and aronia juice were separated using a juicer. The obtained aronia juice was sterilized using a pasteurizer, and then 100% of the aronia juice was concentrated to 60 Brix or more through a low temperature concentrator.

In addition, the pear concentrate was washed after removing impurities from 10,000 kg of pears, and then extracted twice for 3 hours at 95°C at a ratio of pears and distilled water 1:5, and the pear residue was removed through a cylindrical filter with a primary filtration device. Impurities were completely removed using a sediment filter. Subsequently, the concentrator temperature was set at 75° C., and a final concentration of 2,960 kg of 72Brix or more was prepared.

After mixing 10% by weight of Aronia 60Brix obtained above and 10% by weight of pear concentrate 72Brix or more and adding the remaining 80% of water, inoculate 10% by weight of yeast strain ( Saccharomyces cerevisiae ) at 25±2°C in a yeast fermentation tank and fermented for 6 days. Thus, an 8-12% aronia alcohol fermentation broth was prepared. Filtered the Chokeberry alcoholic fermentation stock solution obtained in diatomite 0.45 um filter and then filtered Chokeberry chosangyun primary alcohol fermentation stock solution in acetic acid fermentation tank 30 ± 2 ℃ (Gluconobacter intermedius) 10% inoculation, and 12 days fermentation the pH of more than 5% An acetic acid fermentation broth was prepared.

(3) Preparation of a composition containing red ginseng extract and aronia extract

The prepared red ginseng extract and aronia vinegar are mixed in a weight ratio of 2:1, and as auxiliary ingredients, pear concentrate, liquid fructose, polydextrose, and complex herbal extracts (Astragalus root, cinnamon, Korean Angelica root, Sapju root stem, Dungle, Reishi mushroom) and vitamin C were added and stirred at 75° C. for 50 minutes to prepare a composition including red ginseng extract and aronia extract.

<Test Example 1> Measurement of total polyphenol, flavonoid, and condensed tannin content

(1) Total polyphenol content

Total polyphenol content was determined by the method of Thomas et al. [JH Thomas, et al., J. Sci. Food Agric. 92;2326-2331 (2012)] was modified and measured. Folin-ciocalteu's phenol reagent (Sigma, St. Louis, MO, USA) was added to the samples of red ginseng extract and aronia extract, reacted at room temperature for 3 minutes, and then mixed with 1M Na ₂ CO ₃ solution and reacted for 90 minutes in the dark. Absorbance was measured at 765nm using a plate reader. The phenolic compound content was applied to a standard calibration curve prepared using the standard gallic acid and expressed as gallic acid equivalents (GAE).

(2) total flavonoid content

Analysis of the total flavonoid content was measured using the method of Thomas et al. That is, after mixing 135 µl of distilled water to 25 µl of sample, 10 µl of 5% sodium nitrate was added, reacted for 5 minutes, 15 µl of 10% aluminum chloride was added and reacted for 6 minutes, 50 µl of 1M sodium hydroxide and 50 µl of distilled water After addition, the absorbance was measured at 510 nm. The standard was expressed in mg QE (Qatechin equivalents) using Quercetin.

(3) Condensed tannin content

The total tannin content was measured by modifying the method of Richard et al. (BB Richard, et al., J. Sci. Food Agric. 29;788-794 (1978)). 100 µl of 1% vanillin and 7m H ₂ SO ₄ were added to 50 µl of each extract, followed by mixing, and absorbance was measured at 500 nm after 15 minutes. It was expressed as mg CE (Catechin equivalents) using Catechin.

The results are shown in Table 1 below.

Dilution factor Red ginseng extract and aronia extract X1000 X500 X300 X100 X50 X10 X1 Polyphenols
(GAE) ^a N.D N.D N.D 1.04±0.6 2.50±0.3 12.96±1.4 121.22±10.2 Flavonoids
(QE) ^b 1.94±0.1 2.20±0.1 3.19±0.1 7.02±0.2 10.96±1.0 35.59±6.0 121.19±3.8 Tannins
(CE) ^c N.D N.D N.D N.D N.D 12.11±1.0 115.01±2.4

As shown in Table 1, in the case of red ginseng extract and aronia extract stock solution, the phenol content was 121.22±10.2 µg·GAE/mg, and in the case of flavonoid content, the content of 121.19±3.8 µg·QE/mg was condensed. In the case of tannin content, the content was 115.01±2.4 ㎍·CE/mg. As a result, it is expected to have a positive effect on enhancing antioxidant power. In addition, according to many research reports that flavonoids increase antioxidant activity such as electron donating ability in proportion to the content of phenolic substances, phenolic substances can be said to be an index of verification of the antioxidant effect of natural products.

<Test Example 1> Measurement of antioxidant effect

(1) Measurement of electron donation ability

The electron donating ability (EDA) was measured by applying the method of Thomas et al. In each sample solution 120 µl, 0.45 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) 60 µl was added and allowed to stand for 15 minutes, and the absorbance was measured at 517 nm. As shown in Equation 1 below, the electron donating ability was expressed by the decrease in absorbance of the sample solution-added group and the non-added group.

DPPH assay is a method that can measure a hydrogen donor.It is a method that uses the principle of decolorizing purple by receiving hydrogen or electrons by phenolic compounds, aromatic amines, and ascorbic acid, etc. It is widely used to search for antioxidants. It is widely used because it can measure antioxidant activity in a simple and short time. It was confirmed that the red ginseng extract and aronia extract samples showed more than 90% scavenging ability similar to the positive control (1000 μg/mL) until the condition of 100-fold dilution.

1 shows the measurement results of electron donating ability of red ginseng extract and aronia extract. In FIG. 1, the composition including the red ginseng extract and the aronia extract was referred to as red ginseng aronia stick. As shown here, the red ginseng extract and aronia extract with high total polyphenol and flavonoid content showed relatively high activity, which is consistent with the report that the higher the total polyphenol content, the higher the DPPH radical scavenging activity in the correlation between the total phenol content and the antioxidant activity. It was confirmed that the results showed excellent antioxidant power.

(2) Superoxide dismutase (SOD)-like activity measurement

For SOD-like activity, 130 µl of Tris-HCl buffer (50mM Tris+10mM EDTA, pH 8.5) and 30µl of 7.2mM pyrogallol were added to 40µl of each sample solution, reacted at 37.5℃ for 30 minutes, and then oxidized in the reaction solution at 420nm. The amount of pyrogallol was measured. The SOD-like activity was expressed by the decrease in absorbance of the experimental group and the control group of the sample solution according to Equation 2 below.

SOD enzyme is known as an antioxidant enzyme that removes free radicals in plants and animals, and phenolic compounds such as phenolic acid, flavonoids, anthocyanins and isoflavonoids are known as antioxidants having SOD-like activity. Superoxide dismutase (SOD) is a representative antioxidant enzyme that catalyzes the reaction of converting harmful oxygen radicals formed by oxidation into hydrogen peroxide and converts it into water and oxygen molecules by catalase to protect the living body from active oxygen. .

2 is a result of measuring the superoxide dismutase (SOD)-like activity of red ginseng extract and aronia extract. As shown here, as a result of measuring SOD-like activity, which confirms the scavenging degree of free radicals and peroxides using Pyrogallol, 98% similar to the positive control group (1000 μg/mL) in the stock solution of red ginseng extract and aronia extract samples It was confirmed that it showed abnormal activity. Even when diluted 10 times, it was confirmed that it showed about 30% or more activity.

(3) ABTS cation radical scavenging activity measurement

Antioxidant power measurement using ABTS cation radicals was measured by ABTS cation radical decolorization analysis method. 7mM 2,2-azino-bis(3-ethyl-benthiazoline-6-sulfonic acid) and 2.45mM potassium persulfate are mixed and left at room temperature for 24 hours to form ABTS ⁺ , diluted with ethanol to form ABTS ⁺ 100 µl 100 µl of a sample was added to and left for 7 minutes, and the absorbance was measured at 734 nm.

ABTS radical scavenging activity The ABTS radical (ABTS ^+· ) generated by the reaction of ABTS and potassium persulfate receives electrons by the antioxidant power of the antioxidant material contained in the sample and reduces it to a colorless material. Hydrophobic and hydrophilic sample It is applicable to all.

Figure 3 shows the measurement results of cation radical scavenging ability of red ginseng extract and aronia extract. As shown here, when looking at the ABTS radical scavenging activity, similar to the positive control (1000 μg/mL), ABTS activity was higher than about 98% up to the condition of 100-fold dilution, and showed a similar tendency to the result of electron donating ability. Through these results, samples of red ginseng extract and aronia extract with relatively high phenol and flavonoid content also have excellent ABTS cation radical scavenging ability, which measures cation scavenging ability similar to DPPH radical scavenging ability, which measures anion scavenging ability. It is similar to the results of a study showing that the antioxidant activity increases as the content of total phenolic compounds in the extract sample increases.

(4) Superoxide anion radical scavenging ability measurement

Superoxide anion radical scavenging ability was measured by the nitro blue tetrazolium (NBT) reduction method. To 10 µl of each sample solution and 40 µl of 0.1M potassium phosphate buffer (pH 7.5), 100 µl of a substrate dissolved in xanthine (0.4mM) and NBT (0.24mM) was added, and 100 µl of xanthine oxidase (0.049U/mL) was added. After reacting at 37° C. for 20 minutes, the amount of superoxide anion radical generated in the reaction solution was measured for absorbance at 560 nm.

Xanthine oxidase is a nonspecific enzyme involved in purine metabolism and forms urate from xanthine or hypoxanthine through oxidation. Xanthine oxidase is known to generate hydroxyl radicals (OH-), a kind of ROS, using hypoxanthine as an oxidizing substrate, and the generated large amount of ROS causes pigmentation or skin aging in the skin. In order to evaluate the free radical scavenging ability of red ginseng extract and aronia extract samples, the antioxidant activity was measured using NBT analysis.

Figure 4 shows the measurement results of the superoxide anion radical scavenging ability of red ginseng extract and aronia extract. As shown here, the red ginseng extract and aronia extract samples exhibited more than 70% activity in the stock solution, which was similar to the concentration of 100 μg/mL of the positive control L-ascorbic acid.

(5) FRAP analysis

A 10 mM TPTZ (2,4,6-tripyridyl-S-triazine) solution and 20 mM FeCl _{3 in} acetate buffer (pH 3.6), 40 mM HCl by the method of Iris and Strain (1996) and Yanping et al. (2011) 6H ₂ O was mixed in advance in a ratio of 10:1:1 (v/v/v), respectively, and then heated in a water bath at 37°C. 100 µl of the extract was sequentially mixed and reacted at 37° C. for 4 minutes, and the absorbance was measured at 593 nm using a UV spectrophotometer.

FRAP uses the principle that ferric tripyridyltriazine(Fe ³⁺ -TPTZ) complex is reduced to ferrous tripyridyltriazine (Fe ²⁺ -TPTZ) by a reducing agent at low pH, and is known to have a high dependence on the content of antioxidants in the sample.

Figure 5 shows the measurement results of FRAP analysis of red ginseng extract and aronia extract. As shown here, the OD of iron sulfate hexahydrate (FeSO ₄ ·6H ₂ O) contained in 1000 μg/mL of the sample was determined, and the FRAP values of the red ginseng extract and aronia extract samples were measured. The OD of 2.35±0.02 was similar to that of the positive control group (1000 μg/mL). Iris and Strain (1996) and Yanping et al. (2011) reported that there was a high correlation between the total polyphenol content and the FRAP value.In this study, the total polyphenol content and the FRAP value showed a similar tendency, and the total polyphenol content was not affected by the FRAP value. It is judged to have an effect.

(6) Fe3 ⁺ → Fe2 ⁺ Measuring reducing power

The measurement of reducing power was performed by Gulcin (2007), Gulcin (2010), Elmastas et al. (2016), Bursal er al. (2013) It was measured by modifying the method. To 1 mL of the sample, 200 mM phosphate buffer (pH 6.6) and 1% of potassium ferricyanide were sequentially added and stirred, and then reacted for 20 minutes in a 50°C water bath, and 1 mL of 10% TCA solution was added thereto at 13,500*g. Centrifuged for 15 minutes. Then, 1 mL of distilled water and ferric chloride were mixed in each of the supernatant, and the absorbance was measured at 700 nm.

The reducing power is expressed as the absorbance value of the reducing power converted to ferrous (Fe ²⁺ ) by stabilizing free radicals by donating hydrogen to the ferric-ferricyanide (Fe ³⁺ ) mixture by a substance with reducing power. The stronger the reducing power, the closer to green. It develops color and shows a high absorbance value.

6 shows the measurement results of reducing power of red ginseng extract and aronia extract. The value of L-ascorbic acid (1000 μg/mL) used as a positive control was 2.63±0.02 OD, and the red ginseng extract and aronia extract sample stock solution showed high activity at 2.9±0.08 OD. It has a relatively high reducing power even under conditions, thus exhibiting excellent antioxidant activity.

<Test Example 2> Measurement of anti-inflammatory activity

(1) Macrophage cell line (RAW 264.7) culture method

RAW 264.7 cells, the macrophage cell line used in the experiment, were pre-sold from the Korea Cell Line Bank (KTCC, Seoul, Korea) and used RPMI1640 medium containing 10% FBS (fetalbovine serum), 1% antibiotics, and 2-ME (2-mercaptoethanol). Then, it was cultured in an incubator controlled at 37°C and 4% CO ₂ .

(2) Confirmation of cytotoxicity of test samples

A mouse macrophage line (RAW264.7) was dispensed into a 96-well plate at ⁵ ×10 ⁴ cells/100 μl per well, and then incubated with CO ₂ for 24 hours. One hour after treatment with LPS (lipopolysaccharide) at a concentration of 50 μg/ml, the prepared Aronia red ginseng was diluted in 6 types, treated with 50 μl each, and incubated with CO ₂ for 24 hours. Then, 100 µl of the culture supernatant per condition was removed, and 10 µl of the CCK-8 reagent was treated and incubated for 3 hours with CO ₂ , and absorbance was measured at 450 nm using a microplate reader.

Dilution factor Control LPS Red ginseng extract and aronia extract 1 μg/ml x1000 x100 x10 100.00±0.7 100.04±0.7 97.03±1.8 94.42±1.5 98.58±2.5

7 shows the cytotoxicity of red ginseng extract and aronia extract. As shown here, in the case of the Aronia red ginseng sample, it was confirmed that no cytotoxicity appeared when diluted in 6 ratios.

(3) Measurement of the amount of NO inhibition of macrophage cell line (RAW264.7)

The stabilized NO oxide, NO ₂ (Nitrite), was measured using the Griess reaction. First, a mouse macrophage line (RAW264.7) was dispensed into a 96-well plate at ⁵ ×10 ⁴ cells/100 μl per well, followed by CO ₂ culture for 24 hours. LPS (lipopolysaccharide) was treated with 50 µl each at a concentration of 1µg/ml, incubated with CO2 for 1 hour, and then diluted with 6 types of Aronia red ginseng, added and treated with 50µl, followed by CO2 cultivation for 24 hours. Then, add 50 µl of the culture supernatant per condition to a 96 well plate, and the same amount of Griess reagent (0.1% N-1-naphtyl-ethylendiamine in H ₂ O: 1% sulfanilamide in 5% H ₃ PO ₄ = 1: After 1) was added and reacted for 10 minutes, absorbance was measured at 550 nm with a Microplate reader. The concentration of NO ₂ (Nitrite) was calculated by comparing with the standard curve obtained by diluting NaNO ₂ (sodium nitrite) from 100 μM to 0.7 μM by 2 times.

Dilution factor Control LPS Red ginseng extract and aronia extract 1 μg/ml x1000 x100 x10 0.05±0.1 10.85±0.2 9.36±0.0 8.09±0.2 3.35±0.1

Figure 8 shows the NO inhibitory activity of red ginseng extract and aronia extract. As shown here, as a result of measuring the NO production inhibitory activity, in the case of red ginseng extract and aronia extract, it was confirmed that about 28% inhibition rate was confirmed when diluted X50 times, and when diluted X10 times, 69% NO production was inhibited . LPS was used as a positive control.

(4) Measurement of cytokine production in macrophage cell line (RAW264.7)

RAW264.7 macrophages in a 24-well plate 5X10 ⁵ per well Dispense into cells/1ml and incubate with CO2 for 24 hours. After that, LPS (lipopolysaccharide) was diluted at a concentration of 1 μg/ml and aronia red ginseng in 6 ratios, added to each well, treated with 1 ml, and incubated with CO2 for 24 hours. Then, the cell culture supernatant was collected. The cytokines IL-6, GM-CSF, and IL-1β contained in the supernatant were measured using an enzyme-linked immunosorbent assay (ELISA). That is, dilute the primary antibody (capture antibody) in a plate-bottom micro-well in a coating buffer (0.1 M sodium carbonate, pH 9.5), dispense with 100 µl/well, overnight at 4℃, and wash solution (0.05% Tween). 20/PBS). The washed micro-well was blocked with PBS to which 10% FBS was added, and the culture supernatant collected in the experiment was diluted in an appropriate ratio, and then dispensed into each well and reacted at room temperature. Then, after reacting with 100 µl/well of a secondary antibody to which biotin is attached at room temperature for a certain period of time, 100 µl/well of avidin-peroxidase (enzyme reagent) was added. Finally, a substrate (3,3′,5,5′-Tetramethylbenzidine Liquid Substrate, H ₂ O ₂ ) was added to develop color, and the absorbance was measured at 405 nm using a micro-plate reader. The measured concentrations of IL-6, GM-CSF, and IL-1β were converted using a standard curve.

Dilution factor Cytokine Control LPS
(1 μg/ml) Red ginseng extract and aronia extract X1000 X100 X10 IL-1β N.D 101.36±45.5 65.64±5.1 72.79±5.1 N.D GM-CSF N.D 5888.60±31.6 5687.49±79.0 5380.22±23.7 5117.656±94.8 IL-6 N.D 26.01±0.0 29.82±0.5 30.89±0.7 19.38±0.4

The inflammatory response is induced by an increase in inflammatory cytokines due to an excessive immune response, and representative cytokines include IL-6, IL-1β, and GM-CSF. Among the pro-inflammatory cytokines secreted by LPS in macrophages, IL-6 differentiates B cells into plasma cells and promotes antibody production, transforming the acute inflammatory reaction into a chronic stage. IL-1β acts as a major mediator of the inflammatory response by activating caspase-1 in activated macrophages. In particular, it activates Th cells to induce an excessive inflammatory response. In the case of excessive production of IL-6, sepsis, Alzheimer's, cancer, It is a factor in various diseases such as inflammatory bowel disease.

9 shows the effect of red ginseng extract and aronia extract on inflammatory cytokines (Cytokine). As shown here, as a result of confirming the effect of red ginseng extract and aronia extract sample on the inflammatory cytokine induced by LPS in RAW 264.7 cells, red ginseng extract was similar to the three cytokines of IL-6, IL-1β, and GM-CSF. When the extract and the Aronia extract sample were diluted 10 times, it was confirmed that statistically significant pro-inflammatory cytokine was inhibited.

Claims (4)

A pharmaceutical composition for anti-inflammatory comprising a red ginseng extract and an aronia extract, wherein the aronia extract is a mixture of aronia concentrate and pear concentrate, alcohol fermentation, and then acetic acid fermentation. The method of claim 1,
Anti-inflammatory pharmaceutical composition, characterized in that the red ginseng extract and aronia extract are mixed in a weight ratio of 1:1 to 4:1. A food composition for antioxidant or anti-inflammatory comprising a red ginseng extract and an aronia extract, wherein the aronia extract is a mixture of aronia concentrate and pear concentrate and fermentation with acetic acid after alcohol fermentation. A cosmetic composition for antioxidant or anti-inflammatory comprising a red ginseng extract and an aronia extract, wherein the aronia extract is a mixture of aronia concentrate and pear concentrate, alcohol fermentation, followed by acetic acid fermentation.

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