KR20240088152A - Composition for anti-oxidant, anti-inflammatory, antimicrobial and promotion of digestion comprising Aralia elata extract as effective component and uses thereof - Google Patents

Abstract

The present invention relates to an antioxidant, anti-inflammatory, antibacterial, and digestion-promoting composition containing aralia extract as an active ingredient, and its use. The aralia extract of the present invention has DPPH and ABTS radical scavenging activity, and macrophages induced by inflammatory sources. It is effective in suppressing the production of NO (nitroc oxide) and inflammatory cytokines, antibacterial activity against microorganisms, and enhancing digestive enzyme activity, so it is useful as a health functional food and pharmaceutical composition for antioxidant, anti-inflammatory, antibacterial, or digestion promotion. It can be utilized.

Description

Composition for anti-oxidant, anti-inflammatory, antimicrobial and promotion of digestion comprising Aralia elata extract as effective component and uses thereof}

The present invention relates to an antioxidant, anti-inflammatory, antibacterial and digestion-promoting composition containing aralia extract as an active ingredient and its use.

All living things are known to produce antibacterial and antifungal substances for survival, and research has been attempted for a long time to develop antibacterial or antifungal agents using organisms that produce antibacterial and antifungal substances or substances isolated from them. Antibacterial substances are substances that kill harmful microorganisms, have low toxicity to humans or animals, and have selective toxicity that is not inactivated by enzymes in the body. They mainly involve the replication of DNA, transcription and decoding of genetic information, It is effective through a mechanism that inhibits the growth of microorganisms by inhibiting the transport of electronic energy and cell wall biosynthesis. The main antibacterial substances developed to date can be divided into those of natural origin, such as plants and microorganisms, and those that are chemically synthesized. Many antibacterial substances of natural origin or chemical synthesis are used to treat diseases caused by harmful bacteria, but recently Due to increasing resistance to chemically synthesized antibacterial substances, interest in developing antibacterial agents using naturally derived antibacterial substances is increasing.

In addition, the inflammatory response is a defense response to tissue damage, external physical and chemical stimuli, and various infectious agents, and is a mechanism to repair and regenerate damaged tissues. When an inflammatory reaction occurs, plasma accumulates at the site of inflammation, diluting the toxins secreted by bacteria, blood flow increases, and symptoms such as erythema, pain, swelling, and fever are accompanied. In normal cases, the body neutralizes or eliminates causative factors through an inflammatory response and regenerates damaged tissues to restore normal structure and function. However, a chronic inflammatory response that occurs continuously or excessively causes tissue damage. When an inflammatory response occurs, immune cells such as macrophages and monocytes produce NO (nitric oxide), PGE2 (prostagladin E2), TNF-α (tumor necrosis factor-α), IL-1β (interleukin-1β), IL-6, and IL. Secretes inflammatory mediators such as -8 and IL-12.

Synthetic drugs such as ibuprofen, antihistamines, steroids, cortisone, immunosuppressants, and immune boosters are used to treat inflammation, but the treatment effect is temporary, or side effects such as simple symptom relief, hypersensitivity reactions, and worsening of the immune system occur frequently. Therefore, it is difficult to fundamentally treat inflammation. 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 renal disorders when taken for a long period of time.

Meanwhile, Aralia elata is a shoot that grows on the aralia tree and is a wild vegetable with a unique scent. There are two types of aralia: ground aralia and tree aralia. Ground aralia is the new shoots that sprout from April to May cut out by digging into the ground, and tree aralia is the new shoots that grow on trees. Aralia is rich in protein and contains fat, sugar, fiber, phosphorus, calcium, iron, vitamins (B1, B2, C), saponin, etc. It lowers blood sugar and blood lipids, so it is known to be effective in treating diabetes, kidney disease, or gastrointestinal disease. there is.

Meanwhile, Korean Patent No. 0829832 discloses ‘Cosmetic composition for improving skin wrinkles containing ethanol extract of aralia tree as an active ingredient,’ and Korean Patent No. 0469662 discloses ‘Composition for lowering blood pressure containing extract of aralia tree. ' is disclosed, but there is no description of the antioxidant, anti-inflammatory, antibacterial and digestion-promoting composition containing the aralia extract of the present invention as an active ingredient and its use.

The present invention was developed in response to the above-mentioned needs, and the present inventors confirmed the antioxidant effect of the ethanol extract of aralia by measuring the DPPH and ABTS radical scavenging activity and the total phenol and flavonoid content, and reduced NO (nitroc oxide) production and inflammatory cytokines. The anti-inflammatory effect was confirmed through inhibition of the production of kinesin, TNF-α, IL-6, and IL-1β. In addition, the ethanol extract of aralia has an antibacterial effect against Salmonella typhimurium , Listeria monocytogenes, and Helicobacter pylori , and the digestive enzymes α-amylase and protease. ), the present invention was completed by confirming that it has a digestion-promoting effect by increasing the activity of.

In order to solve the above problems, the present invention provides an antioxidant, anti-inflammatory, antibacterial, and digestion-promoting health functional food composition containing aralia extract as an active ingredient.

Additionally, the present invention provides an antibacterial and anti-inflammatory pharmaceutical composition containing aralia extract as an active ingredient.

Additionally, the present invention provides an antibacterial and anti-inflammatory veterinary composition containing aralia extract as an active ingredient.

Additionally, the present invention provides an antioxidant, anti-inflammatory, antibacterial, and digestion-promoting feed additive containing aralia extract as an active ingredient.

The aralia extract of the present invention has excellent DPPH and ABTS radical scavenging activity, inhibitory activity on NO and inflammatory cytokine production in macrophages induced by inflammatory sources, antibacterial activity against microorganisms, and an effect of increasing digestive enzyme activity, and is therefore an antioxidant. , It can be useful as a health functional food and pharmaceutical composition for anti-inflammatory, antibacterial, or digestion promotion.

Figure 1 shows the results of high performance liquid chromatography (HPLC) on a chlorogenic acid standard solution (A) and an aralia ethanol extract (B).

In order to achieve the object of the present invention, the present invention provides an antioxidant, anti-inflammatory, antibacterial and digestion-promoting health functional food composition containing aralia extract as an active ingredient.

In the health functional food composition of the present invention, the aralia extract may be extracted using water, a lower alcohol of C ₁ to C ₄ , or a mixture thereof as a solvent, preferably using ethanol. It is not limited to this.

In one embodiment of the present invention, the aralia extract contains a high content of total polyphenols and flavonoids, has excellent antioxidant activity, and reduces NO and inflammatory cytokines (TNF-α, IL) in LPS-treated macrophages. -6 and IL-1β) production, it has excellent anti-inflammatory effects and has excellent antibacterial effects against Salmonella typhimurium, Listeria monocytogenes, and Helicobacter pylori . It is characterized by an excellent digestion-promoting effect by increasing the activity of digestive enzymes α-amylase and protease.

The active ingredient of the present invention can be added directly to food or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention or improvement). In general, the content of the active ingredient contained in the health functional food can be added in the range of 0.1 to 90 parts by weight of the total weight of the health functional food. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

The health functional food composition of the present invention may be in a formulation selected from powder, granule, pill, tablet, capsule, candy, syrup, and beverage, but is not limited thereto.

There are no particular restrictions on the types of health functional foods. Examples of foods to which the health functional food composition can be added include meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, and tea. There are drinks, alcoholic beverages, and vitamin complexes, and it includes all health foods in the conventional sense.

Additionally, the health functional food composition of the present invention can be manufactured into food, especially functional food. The functional food of the present invention includes ingredients commonly added during food production, and includes, for example, proteins, carbohydrates, fats, nutrients and seasonings. For example, when manufacturing a drink, natural carbohydrates or flavoring agents may be included as additional ingredients in addition to the active ingredient. The natural carbohydrates include monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g., dextrins, cyclodextrins, etc.), or sugar alcohols (e.g., For example, xylitol, sorbitol, erythritol, etc.) is preferable. The flavoring agent may be a natural flavoring agent (e.g., thaumatin, stevia extract, etc.) or a synthetic flavoring agent (e.g., saccharin, aspartame, etc.).

In addition to the above health functional food composition, various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonic acid. It may further contain carbonating agents used in beverages. The ratio of the ingredients added is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food composition of the present invention.

The present invention also provides an antibacterial and anti-inflammatory pharmaceutical composition containing aralia extract as an active ingredient.

In the pharmaceutical composition of the present invention, the aralia extract is as described above.

The pharmaceutical composition containing the aralia extract of the present invention may further include an appropriate carrier, excipient, or diluent commonly used in the preparation of pharmaceutical compositions.

The pharmaceutical dosage form of the pharmaceutical composition of the present invention can be used alone or in combination with other pharmaceutically active compounds, as well as in appropriate combinations.

The pharmaceutical composition according to the present invention can be formulated and used in the form of oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and injections according to conventional methods. . Carriers, excipients, and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, and cellulose. , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain the active ingredients plus at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. It is prepared by mixing. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, and syrups. In addition to the 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. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, wethepsol, macrogol, Tween 61, cacao, laurin, glycerogelatin, etc. can be used.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, degree of disease, drug form, administration route and period, but can be appropriately selected by a person skilled in the art. The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, and humans through various routes. All modes of administration are contemplated, for example, oral, rectal or by intravenous, intramuscular, subcutaneous, intrathecal or intracerebroventricular injection.

Additionally, the present invention provides an antibacterial and anti-inflammatory veterinary composition containing aralia extract as an active ingredient.

The veterinary composition of the present invention may further include appropriate excipients and diluents according to conventional methods. Excipients and diluents that may be included in the veterinary composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate, cetanol, stearyl alcohol, liquid paraffin, sorbitan monostearate. , polysorbate 60, methylparaben, propylparaben, and mineral oil. The veterinary composition according to the present invention may further include fillers, anti-aggregants, lubricants, wetting agents, spices, emulsifiers, preservatives, etc. The veterinary composition according to the present invention provides rapid and sustained release of the active ingredient after administration to an animal. or may be formulated using methods well known in the art to provide sustained release, the dosage form being powders, granules, tablets, capsules, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, It may be in the form of a suppository, sterile injectable solution, or sterile topical medication.

The effective amount of the veterinary composition according to the present invention can be appropriately selected depending on the individual animal. Severity of the disease or condition, sensitivity to the active ingredient of the present invention depending on the individual's age, weight, health condition or gender, administration route, administration period, factors including other compositions mixed or used simultaneously with the composition, and other physiological or It can be determined based on factors well known in the veterinary field.

Additionally, the present invention provides an antioxidant, anti-inflammatory, antibacterial, and digestion-promoting feed additive containing aralia extract as an active ingredient.

The feed additive of the present invention corresponds to supplementary feed under the Feed Management Act. In the present invention, the term 'feed' may mean any natural or artificial diet, meal, etc., or a component of the meal, for or suitable for eating, ingestion, and digestion by animals. The type of feed is not particularly limited, and feed commonly used in the art can be used. Non-limiting examples of the feed include plant feeds such as grains, roots and fruits, food processing by-products, algae, fiber, pharmaceutical by-products, oils and fats, starches, cucurbits or grain by-products; Examples include animal feeds such as proteins, inorganic substances, fats and oils, minerals, fats and oils, single-cell proteins, zooplanktons or foods. These may be used alone or in combination of two or more types.

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

Materials and Methods

1. Preparation of aralia extract

Aralia produced in Sunchang, Jeollabuk-do was purchased at the Sunchang-gun agricultural specialty product direct sales store and dried using a freeze dryer (LCLN-50C, Nihon freezer, Japan). Completely dried aralia was pulverized using a grinder (HR3752/00, Philips, Netherlands), then mixed with aralia powder and 70% ethanol in a 1:5 ratio and extracted for 4 hours at 25°C. It was centrifuged at 3,000 The filtrate was concentrated to 30 brix using a vacuum concentrator (N-1000, Eyela, Japan), then completely dried using a freeze dryer to form powder (3.16 g, 15.8% yield), stored at -20°C, and used in the experiment. .

2. Measurement of chlorogenic acid content

To measure the chlorogenic acid content in aralia extract, 50 ml of methanol (Burdick & Jackson Co., Muskegon, USA) was added to 1 g of aralia extract, followed by ultrasonic extraction for 30 minutes, followed by 0.45 μm syringe filter (Whatman Co., UK). It was filtered and used as an analysis sample.

Chlorogenic acid (Sigma Chemical Co., USA) standard solution was prepared at a concentration of 1 mg/ml using methanol and analyzed by filtering through a 0.2 μm syringe filter. The HPLC analysis device is an HPLC system (Agilent 1200 series, Agilent Technologies, USA), the DAD (Diode array detector) is G1315D 1200 series (254nm, Agilent Technologies), and the column is ZORBAX Eclipse Plus C18 (4.6×250 mm, 5 μm, Agilent). Technologies) was used. The mobile phases were formic acid (A) and methanol (MeOH, B), the flow rate was 1.0 mL/min, and the column temperature was set to 25°C for analysis (Table 1). All solvents were degassed and filtered before use, and the quantitative curve of chlorogenic acid was analyzed by drawing peak area ratio and concentration graphs.

HPLC conditions Instrument HPLC system (Agilent, 1200 series) Detector Diode array detector(DAD G1315D) (254nm) Column Agilent ZORBAX Eclipse Plus C18 (4.6×250 mm, 5 μm) Temperature 25℃ Flow rate 1.0 ㎖/min Injection volume 20 μl Mobile phase Time (min) Water Acetonitrile
0 90 10
7 75 25
17 65 35
25 50 50
30 20 80 Run time 30min

3. Measurement of antioxidant activity

To measure DPPH radical scavenging activity, 40 ㎕ of aralia extract at a concentration of 312.5~10,000 ㎖/㎖ and 180 ㎕ of 0.2 mM DPPH solution were dispensed into each well of a 96-well plate, reacted at 37°C for 30 minutes, and then incubated with a microplate reader. Absorbance was measured at 515 nm using (Infinite Pro 200, Tecan, Austria). DPPH radical scavenging activity was calculated using the formula below, and was expressed as a scavenging concentration (SC) ₅₀ , which is the concentration that reduces DPPH radicals by 50%. L-ascorbic acid was used as a control.

DPPH radical scavenging activity (%) = 1-(absorbance of sample-added group/absorbance of sample-free group) × 100

Additionally, to measure ABTS radical scavenging activity, 2.4 mM potassium persulfate was mixed with 7 mM ABTS solution, reacted for 12 hours, and then adjusted using distilled water so that the absorbance at 734 nm was 1.0. 10 μl of sample and 190 μl of ABTS solution were dispensed into each well of a 96-well plate, reacted at room temperature for 10 minutes, and absorbance was measured at 734 nm using a microplate reader. ABTS radical scavenging activity was calculated using the formula below and expressed as the concentration (SC ₅₀ ) that reduces ABTS radicals by 50%, and L-ascorbic acid was used as a control.

ABTS radical scavenging activity (%)=1-(absorbance of sample added group/absorbance of sample-free group)×100

4. Determination of total polyphenol and total flavonoid content

To measure the total polyphenol content, 0.5 ml of 1 N Folin-Ciocalteu's reagent and 1 ml of 5% Na ₂ CO ₃ were added to 1 ml of aralia extract, reacted in the dark for 1 hour, and then measured using a UV-Vis spectrophotometer. Absorbance was measured at 725 nm using Agilent 8453 (Agilent, USA). The total polyphenol content was measured using the standard curve of gallic acid (25-200 ㎕/㎖, regression equation y=0.0055x-0.0318, R ² =0.9969), and the total polyphenol content was calculated as mg gallic acid equivalent. Expressed as (GAE)/g.

To measure the total flavonoid content, 0.1 ml of 10% aluminum nitrate solution, 0.1 ml of 1 M potassium acetate solution, and 4.3 ml of ethanol were added to 0.5 ml of aralia extract and reacted at room temperature for 40 minutes. Then, the absorbance was measured at 415 nm using a spectrophotometer. The total flavonoid content was measured using the standard curve of quercetin (20-100 ㎕/㎖, regression equation y=0.0014x+0.0011, R ² =0.9989), and the total flavonoid content was calculated as mg quercetin equivalent (QE)/ Expressed in g.

5. Cytotoxicity measurement

To measure cytotoxicity against RAW 264.7 cells and human colorectal cancer cell line (Caco-2), RAW 264.7 cells were distributed at 1×10 ⁵ cells/well in a 96-well plate and incubated at 37°C in a 5% CO ₂ incubator. After culturing for 24 hours, the aralia extract was treated at different concentrations and cultured in a CO ₂ incubator for 24 hours. Cultured cells were treated with 0.5 mg/ml of MTT reagent and reacted for 4 hours, the supernatant was removed, and the resulting formazan was completely dissolved in 100 ㎕ of DMSO, and the absorbance was measured at 540 nm using a spectrophotometer. did. In addition, Caco-2 cells were distributed in a 96-well plate at 2.5 × 10 ⁵ cells/well, cultured in a 37°C, 5% CO ₂ incubator for 24 hours, and then measured in the same manner as above.

6. Measurement of NO (nitric oxide) production inhibition

To measure NO produced from RAW 264.7 cells and Caco-2 cells, RAW 264.7 cells were distributed at 1 × 10 ⁵ cells/well in a 24-well plate and cultured in an incubator at 37°C and 5% CO ₂ for 24 hours. Afterwards, the aralia extract was pretreated for 2 hours, treated with LPS (1 μg/ml) to induce NO production, and cultured in a 37°C, 5% CO ₂ incubator for 24 hours. Afterwards, the culture medium was reacted with Griess reagent (0.1% N-(1-naphtyl) ethylenediamine: 1% sulfanilamide = 1:1), and the absorbance was measured at 570 nm using a spectrophotometer. Sodium nitrate was diluted, absorbance was measured, and the NO production inhibition effect was evaluated using a standard curve. In addition, Caco-2 cells were distributed in a 24-well plate at 2.5 × 10 ⁵ cells/well and cultured at 37°C in a 5% CO ₂ incubator for 48 hours, and then measured in the same manner as above.

7. Measurement of inflammatory cytokine secretion

RAW 264.7 cells were distributed in a 24-well plate _at ⁵ After treatment and culturing for 24 hours, the supernatant was collected and used in the experiment. For inflammatory cytokines, the production of TNF-α, IL-1β, and IL-6 was measured using the ELISA MAXTM Deluxe Set ELISA kit (BioLegend, USA) according to the manufacturer's instructions.

8. Measurement of antibacterial activity

Listeria monocytogenes and Antibacterial activity against Salmonella typhimurium was measured by creating a clear zone using agar well diffusion and paper disc diffusion, and the minimum growth inhibition concentration (MIC) was measured. ) and minimum bactericidal concentration (MBC) were measured using the Broth-microdilution method.

Each strain was inoculated into LB broth supplemented with aralia extract, cultured at 37°C for 24 hours, absorbance was measured at 600 nm, and MIC was measured based on growth changes of the strain. To confirm the antibacterial activity of the aralia extract and measure the potency of the antibacterial activity, MBC was determined by measuring the number of viable cells. Aralia extract was added to the strain culture medium at a concentration of 400 mg/ml, which is higher than the MIC, and applied to LB agar medium and cultured for 24 hours to measure the number of viable bacteria. The minimum concentration at which the number of killed bacteria exceeded 99.9% was determined as MBC. After uniformly dispensing Brucella broth containing aralia extract, Helicobacter pylori The strain was inoculated at 0.5×10 ⁷ CFU/ml and cultured in a 10% CO ₂ incubator at 37°C for 24 hours. The MIC and MBC concentration were determined by measuring the growth changes and degree of death of the strain using the same method as above.

9. Measurement of digestive enzyme activity

To measure α-amylase enzyme activity, 500 ㎕ of aralia extract was mixed with 250 ㎕ of 1.0% (w/v) Soluble starch solution (20 mM sodium phosphate buffer, 6.7 mM sodium chloride, pH 6.9) and incubated at 37°C. After reacting for 30 minutes, 0.5 ㎕ of 96mM DNS (dinitrosalicylic acid) solution was added and reacted at 100°C for 5 minutes, then left at 4°C for 3 minutes and absorbance was measured at 540 nm. The standard curve was prepared using 0.2% (w/v) maltose, and the enzyme activity (unit definition) was 1.0 μg produced from starch in 1 minute under the above reaction conditions (37°C, pH 6.9). The amount of maltose was defined as 1 unit.

Additionally, to measure protease enzyme activity, 0.5 ml of aralia extract was added to 1.25 ml of 0.65% (w/v) casein buffer (casein 6.5 g/ml, 50 mM potassium phosphate buffer, pH 7.5) and After reacting at ℃ for 10 minutes, the reaction was stopped by adding 2.5 ml of 110 mM trichloroacetic acid. After the reaction sample was left standing at 37°C for 30 minutes, the remaining precipitate was filtered through a 0.45 μm syringe filter. 5 ml of 500 mM sodium carbonate solution and 1 ml of 0.5 M Folin & Ciocalteu's phenol reagent were mixed with 2 ml of the filtrate, reacted at 37°C for 30 minutes, and absorbance was measured at 660 nm. The standard curve was created by analyzing the same method as above using L-tyrosine, and 1 unit was calculated by converting the amount of 1 μg of tyrosine released in 1 minute.

10. Statistical processing

For statistical processing of all experiments, mean ± standard deviation was calculated using the Sigma plot (sigma plot for windows version 10.0, USA) program. For analysis between two groups, Student's t -test was performed, and for analysis between three or more groups, one-way ANOVA and Tukey's multiple comparison test were performed as a post hoc analysis. p <0.05 was interpreted as significant.

Example 1. Analysis of chlorogenic acid content in aralia extract

The chlorogenic acid content contained in the ethanol extract of aralia was converted to the peak area consistent with the retention time of the chlorogenic acid standard solution. In the chlorogenic acid standard solution, the largest peak was observed at a retention time of 13.01 minutes (Figure 1A), and in the aralia ethanol extract (Figure 1B), a peak was observed at a retention time of 13.00 minutes, similar to the chlorogenic acid standard solution. In addition, the chlorogenic acid content in the aralia extract was confirmed to be 7.06 ± 0.01 mg/g.

Example 2. Analysis of antioxidant activity of aralia extract

2-1. DPPH and ABTS radical scavenging activity

As a result of measuring the SC ₅₀ value of the aralia ethanol extract (hereinafter referred to as AEE), which scavenges 50% of DPPH radicals, and the SC ₅₀ value of the positive control group, AEE was 4.79 ± 0.05 g/ml, and the positive control group (L-ascorbic acid, hereinafter referred to as L-ascorbic acid) was measured. AA) was confirmed to be 0.19±0.00 g/ml.

In addition, as a result of measuring the SC ₅₀ value of AEE, which scavenges 50% of ABTS radicals, and the SC ₅₀ value of the positive control group, it was confirmed that AEE was 5.79 ± 0.05 g/ml and AA was 0.11 ± 0.00 g/ml (Table 2).

DPPH and ABTS radical scavenging activity measurement results Sample DPPH radical scavenging ABTS radical scavenging SC ₅₀ (mg/ml) SC ₅₀ (mg/ml) AEE 4.79±0.05 5.79±0.05 AA 0.19±0.00 0.11±0.00

2-2. Total polyphenol and total flavonoid content

As a result of measuring the total polyphenol and total flavonoid contents in AEE, it was confirmed that they were 170.0 ± 1.8 mg GAE/g and 105.4 ± 4.1 mg QE/g, respectively.

Example 3. Analysis of anti-inflammatory effect of aralia extract

Through the MTT assay, AEE was administered to RAW 264.7 macrophages and Caco-2 cells at different concentrations (25 μg/ml, 50 μg/ml, 75 μg/ml, 100 μg/ml, 125 μg/ml, 150 μg/ml, As a result of measuring cell viability by treatment with 200 μg/ml or 250 μg/ml), RAW 264.7 cells showed a survival rate of more than 100% up to a concentration of 150 μg/ml, and Caco-2 cells showed a survival rate of more than 100% up to a concentration of 250 μg/ml. It showed a survival rate of more than %. Through this, the concentration of aralia extract was set to 100 μg/ml or less in measuring NO production and secretion of inflammatory cytokines.

In addition, as a result of measuring the amount of NO production in RAW 264.7 cells and Caco-2 cells, it was confirmed that the amount of NO production decreased in a concentration-dependent manner in the AEE-treated group compared to the LPS-only treated group (untreated with aralia extract) (Table 3). It was confirmed that the production of inflammatory cytokines TNF-α, IL-6, and IL-1β in RAW 264.7 cells was also decreased in a concentration-dependent manner (Table 4).

NO production measurement results Cell line Control LPS
(1 ㎍/㎖) Concentration (㎍/㎖) + LPS (1㎍/㎖) 25 50 75 100 Raw 264.7 cells 1.9± ^0.3z 100.0± ^0.0y 79.3± ^1.7x 73.9± ^0.9w 65.5± ^0.3V 60.0± ^1.1u Caco-2 cells 0.9± ^0.1z 100.0± ^0.0y 63.9± ^1.3x 59.7± ^0.3w 56.4± ^1.7V 50.7± ^2.8u

Inflammatory cytokine production measurement results Cytokines Control LPS
(1 ㎍/㎖) Concentration (㎍/㎖) + LPS (1㎍/㎖) 25 50 75 100 TNF-α
(ng/ml) 2.5± ^0.0z 11.9± ^0.3y 11.4± ^0.1x 10.4± ^0.3V 9.3± ^0.3u 8.9± ^0.1u IL-6
(ng/ml) 0.4± ^0.0z 24.0± ^0.2y 18.2± ^0.1x 16.8± ^0.2V 15.2±0.2 ^u 15.2± ^0.8u IL-1β
(pg/ml) 24.9± ^3.5z 75.5±4.7 ^y 36.9± ^2.9x 36.4± ^0.7wd 35.5±1.0 ^wv 30.9± ^0.9V

Example 4. Analysis of antibacterial effect of aralia extract

Salmonella typhimurium ( S. typhimurium ) and Listeria mono of AEE at different concentrations (1 mg/disc, 2 mg/disc, 3 mg/disc) using agar well diffusion and paper disc diffusion. As a result of measuring the antibacterial activity against L. monocytogenes and Helicobacter pylori ( H. pylori ), it was confirmed that the size of the clear zone increased in a concentration-dependent manner of AEE for all strains (Table 5) .

Antibacterial activity measurement results Bacteria Diffusion Assay Diameter of clean zone (㎜) Concentration (㎎/disc) One 2 3 S. Typhimurium Agar well N.D. N.D. 0.64 Paper disc ^{0.01z 0.02z 0.57y} L. monocytogenes Agar well ^{1.32z 1.86y} 3.99 ^x Paper disc N.D. ^{2.10z 2.17y} H. pylori Agar well ^{1.56z 4.46y 6.10x} Paper disc 2.49 ^{z 6.67y 7.14x}

In addition, as a result of AEE measuring the minimum concentration (MIC) and minimum killing concentration (MBC) that can inhibit the growth of Salmonella Typhimurium, Listeria monocytogenes, and Helicobacter pylori, the MIC of Salmonella Typhimurium and Helicobacter pylori were It was confirmed that the MIC of Listeria monocytogenes was 100 mg/ml and MBC was 350 mg/ml, and the MIC of Listeria monocytogenes was 75 mg/ml and MBC was 275 mg/ml.

Example 5. Analysis of the digestion-promoting effect of aralia extract

As a result of measuring the α-amylase and protease activities of AEE at different concentrations, α-amylase activity increased up to a concentration of 20 mg/ml and tended to decrease slightly at 25 mg/ml, and protease activity increased depending on the concentration of AEE. It was confirmed that (Table 6).

Claims (6)

A health functional food composition containing aralia extract as an active ingredient for antioxidant, anti-inflammatory, antibacterial and digestion promotion purposes. The method of claim 1, wherein the aralia extract inhibits NO (nitroc oxide) production and the production of inflammatory cytokines TNF-α, IL-6, and IL-1β, and inhibits the digestive enzymes α-amylase and protease. ) A health functional food composition characterized by increasing the activity of. The method of claim 1, wherein the aralia extract exhibits antibacterial activity against one or more bacteria selected from the group consisting of Salmonella typhimurium, Listeria monocytogenes, and Helicobacter pylori. A health functional food composition made of. An antibacterial and anti-inflammatory pharmaceutical composition containing aralia extract as an active ingredient. An antibacterial and anti-inflammatory veterinary composition containing aralia extract as an active ingredient. An antioxidant, anti-inflammatory, antibacterial, and digestion-promoting feed additive containing aralia extract as an active ingredient.