KR102161319B1 - An anti-inflamatory composition comprising solid-state-fermented Caryopteris incana extract as an active ingredient and preparation method thereof - Google Patents

Abstract

The present invention relates to an anti-inflammatory composition and a method for preparing the same, and a composition for anti-inflammatory containing as an active ingredient a dogwood extract, which is fermented with lactic acid bacteria ( Lactobacillus plantarum ) and has an increased anti-inflammatory effect, and the present inventors have a layer fermented with lactic acid bacteria ( Lactobacillus plantarum ). By reducing the expression of lipopolysaccharide (LPS)-induced inflammatory mediators in Raw 264.7 cells, flower tree extract effectively suppressed the inflammatory response of cells, and it was confirmed that the cytotoxicity was lower than that of non-fermented dogwood extract. Therefore, by establishing the fermentation system according to the present invention, it is expected that the toxicity of dogwood trees can be reduced and the anti-inflammatory activity can be increased to secure excellent herbal resources with high productivity of pharmacologically active compounds, and solid fermented dogwood extract is a new anti-inflammatory agent. It can be usefully used as an excellent material for development and a health functional food composition for improving inflammation.

Description

An anti-inflamatory composition comprising solid-state-fermented Caryopteris incana extract as an active ingredient and preparation method thereof, containing solid fermented dogwood fermented extract with lactic acid bacteria as an active ingredient thereof}

The present invention relates to a composition for anti-inflammatory containing as an active ingredient a fermented dogwood fermented extract having an increased anti-inflammatory effect by being solid-fermented with lactic acid bacteria ( Lactobacillus plantarum ) and a method for preparing the same.

The inflammatory reaction is a defense reaction of biological tissues against external stimuli, and is a mechanism to regenerate damage caused by physical and chemical stimuli, harmful substances and bacterial infections. In normal inflammatory reactions, antigens are removed by antigen-antibody reactions, but in chronic inflammatory reactions, certain tissues are damaged and various diseases such as atopy, arthritis, and psoriasis are caused. In the immune system, macrophages regulate immune function by suppressing inflammatory reactions expressed by various stimuli such as free radicals and stress, and play a very important role in maintaining homeostasis. In macrophages, stimulation such as cytokines, tumar necrosis factor (TNFα), and lipopolysaccharide (LPS) activates nuclear factorκB (NFκB), a transcription factor for inflammatory responses, and as a result, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX2) By expressing an excess of nitric oxide (NO) and prostaglandin E2 (PGE2), causing inflammation. Synthetic anti-inflammatory drugs developed so far can be largely divided into steroidal (glucocorticoid) and nonsteroidal (ibuprofen, diclofenac). Most of them have side effects on digestive tract disorders and heart disease, making the development of safer and more effective natural anti-inflammatory drugs. It is an urgent situation.

The dogwood (Caryopteris incana) is a perennial plant and is widely distributed in Korea, China, Taiwan, and Japan, and in Korea, it mainly lives in mountains and fields. The stem, 30~60 cm tall, is upright and the base is woody, and the opposite leaves are long oval, 2.5~6 cm long, 1.5~3 cm wide, and flowers bloom purple or white in July-September. The inflorescences are arranged in layers, and the black fruits open as capsules from September to October. Young sprouts are eaten as herbs and are used as herbal medicinal herbs. Herb or root is medicated, and the herbal name is Nanhyangcho (蘭香草). The taste is spicy and the yakseong is warm. The outpost of the dogwood tree is effective in anti-oxidation, anti-cancer and diabetes, and flavonoid glycosides and phenols are known to inhibit allergic reactions, alkaloids, steroids, and amino acids known to have vasodilating effects. (aminoacid), organic acid with proven antibacterial effect, tannin, which has astringent taste and astringent effect, contains astringent, cough, phlegm, bronchitis, neuralgia, boils, menstrual impurities, blood stagnation, bruises, eczema, skin itching It is used for the treatment of back, and is known to have antibacterial activity against Staphylococcus aureus and Corynebacterium diphtheriae .

Meanwhile, in general, the fermentation process of food has beneficial effects such as increasing useful ingredients, imparting new physiological activity, increasing absorption rate, reducing residual pesticides, and increasing useful intestinal microbes. The biological conversion method used is being used in the product. However, until now, there has been no report on a fermentation method that enhances the anti-inflammatory effect of dogwood.

Korean Patent Registration No. 10-1321879

Accordingly, as a result of the present inventors making diligent efforts to increase the inhibitory effect of inflammation of the dogwood , not only the cytotoxicity of the solid fermented dogwood ethanol extract with lactic acid bacteria ( Lactobacillus plantarum ) was lower than that of the non-fermented dogwood ethanol extract, By reducing the expression of inflammatory mediators, it was confirmed that the inflammatory response of cells was effectively suppressed, and a lactic acid bacteria fermentation technique was developed to increase the functional activity of dogwood.

Accordingly, an object of the present invention is to provide an anti-inflammatory pharmaceutical composition containing a fermented dogwood fermented extract as an active ingredient.

In addition, another object of the present invention is to provide a health functional food composition for improving inflammation containing a fermented dogwood fermented extract as an active ingredient.

In addition, another object of the present invention is to provide a method for producing a fermented dogwood fermented extract.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention provides an anti-inflammatory pharmaceutical composition containing a fermented dogwood fermented extract as an active ingredient.

In addition, the present invention provides a health functional food composition for improving inflammation containing a fermented dogwood fermented extract as an active ingredient.

In addition, the present invention provides a method for producing a fermented dogwood fermented extract.

As an embodiment of the present invention, the fermented dogwood extract is characterized in that it is fermented with lactic acid bacteria ( Lactobacillus plantarum ).

In another embodiment of the present invention, the extract is characterized in that the ethanol extract, the concentration of the ethanol may be 80% by weight, but is not limited thereto.

In another embodiment of the present invention, the fermented dogwood fermented extract is characterized in that it exhibits an effect of inhibiting the expression of inducible nitrogen oxide synthase (iNOS).

In another embodiment of the present invention, the fermented dogwood fermented extract is characterized in that it exhibits an effect of inhibiting the expression of cyclooxygenase (COX2).

As another embodiment of the present invention, the fermented dogwood fermented extract is prostaglandin E ₂ (PGE ₂ ) It is characterized by exhibiting a production inhibitory effect.

In another embodiment of the present invention, the fermented dogwood fermented extract is characterized in that it exhibits an effect of inhibiting the expression of cyclooxygenase (COX2).

In another embodiment of the present invention, the fermented dogwood extract is any one or more cytotoxics selected from the group consisting of tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and interleukin-1β (IL-1β). It is characterized by exhibiting an inhibitory effect on the expression of kine (Cytokine).

Furthermore, the present invention provides a method for treating inflammation comprising administering a pharmaceutical composition for anti-inflammatory containing a fermented dogwood fermented extract as an active ingredient to an individual in need of treatment for inflammation.

In addition, the present invention provides an anti-inflammatory pharmaceutical composition containing a fermented dogwood extract as an active ingredient for the treatment of inflammation.

The anti-inflammatory composition containing the solid fermented extract of P. serrata of the present invention as an active ingredient is fermented with lactic acid bacteria ( Lactobacillus plantarum ), by reducing the expression of lipopolysaccharide (LPS)-induced inflammatory mediators in Raw 264.7 cells. It effectively inhibits the inflammatory reaction and has low cytotoxicity compared to non-fermented dogwood extract. Therefore, by establishing the fermentation system according to the present invention, it is expected that the toxicity of the dogwood tree can be reduced and the anti-inflammatory activity can be increased, thereby securing excellent herbal resources with high productivity of the pharmacologically active compound. It can be usefully used as an excellent material for and a health functional food composition for improving inflammation.

1 is a view showing a result of measuring the elution amount of a phenolic compound by extracting dogwood with various solvents and concentrations.
2 is a view showing the result of measuring the elution amount of the phenolic compound of the dogwood extract extracted by setting the concentration of ethanol step by step.
Figures 3a and 3b are views showing the results of confirming the cytotoxicity of the non-fermented dogwood ethanol extract (Figure 3a) and fermented ethanol extract (Figure 3b).
4A and 4B are diagrams showing the results of measuring the expression level of iNOS protein in the non-fermented dogwood ethanol extract (FIG. 4a) and fermented ethanol extract (FIG. 4B).
5A and 5B are diagrams showing the results of measuring the expression level of COX2 protein of the non-fermented dogwood ethanol extract (FIG. 5A) and fermented ethanol extract (FIG. 5B).
6a and 6b are views showing the results of measuring the amount of NO in the non-fermented dogwood ethanol extract (FIG. 6a) and fermented ethanol extract (FIG. 6b).
7a and 7b are diagrams showing the results of measuring the amount of PGE ₂ produced by the non-fermented dogwood ethanol extract (FIG. 7a) and fermented ethanol extract (FIG. 7b ).
8A and 8B are diagrams showing the results of measuring the expression level of TNFα in the ethanol extract of non-fermented dogwood (FIG. 8a) and the fermented ethanol extract (FIG. 8B).
9A and 9B are diagrams showing the results of measuring the expression level of IL6 in the non-fermented dogwood ethanol extract (FIG. 9a) and fermented ethanol extract (FIG. 9B).
10A and 10B are diagrams showing the results of measuring the expression level of IL1β in the non-fermented dogwood ethanol extract (FIG. 10a) and fermented ethanol extract (FIG. 10B).

The present inventors layer flowering trees lactic acid powder (Lactobacillus plantarum), after the solid fermentation by reducing the expression of inflammatory mediators, as well as the cytotoxicity lower than the layer flowering trees ethanol extract is prepared layer flowering trees fermentation extract non-fermented cells It was confirmed that it effectively inhibits the inflammatory reaction, and the present invention was completed based on this.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for anti-inflammatory containing the fermented dogwood fermented extract as an active ingredient.

In addition, the present invention provides a health functional food composition for improving inflammation containing a fermented dogwood fermented extract as an active ingredient.

In addition, the present invention provides a method for producing a fermented dogwood fermented extract.

In an embodiment of the present invention, a fermented dogwood fermented extract was prepared (see Example 1), and the elution amount of a phenolic compound according to a change in solvent type and concentration of the dogwood extract was compared (see Example 2).

In another embodiment of the present invention, the anti-inflammatory effect of the non-fermented ethanol extract and the fermented ethanol extract of dogwood was compared (see Example 3).

In another embodiment of the present invention, the non-fermented ethanol extract and the fermented ethanol extract of the cytokine including tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and interleukin-1β (IL-1β) The inhibitory activity of was confirmed (see Example 4).

Therefore, in light of the above results, it is expected that excellent herbal resources with high productivity of pharmacologically active compounds can be secured through the dogwood extract fermented with lactic acid bacteria according to the present invention.

The dogwood extract of the present invention may be prepared by using a conventional solvent under a conventional method known in the art, that is, under the conditions of a conventional temperature and pressure. Extraction solvents that can be used to prepare the dogwood extract of the present invention include, for example, water, C1 to C4 lower alcohol, n-hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride. , And an extraction solvent such as a mixed solvent thereof may be used alone or in combination, and it is preferably ethanol, but is not limited thereto. The appropriate amount of the extraction solvent is about 1 to 10 times the dry weight of the dogwood, and as an extraction method, heat extraction, cold needle extraction, reflux cooling extraction, ultrasonic extraction, etc. can be used, and the extraction is repeated once or multiple times. Can be used. Further, the extraction temperature is not particularly limited as long as it is a temperature at which the effective activity of the useful components of the dogwood is not removed.

The composition of the present invention may contain one or more known active ingredients having an inflammatory treatment effect together with the dogwood extract.

As used herein, the term "administering" means providing a given composition of the present invention to an individual in any suitable manner.

The composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. Pharmaceutically acceptable carriers can be used by mixing saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these ingredients, and if necessary, antioxidants and buffers , Other conventional additives such as bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare injectable formulations such as aqueous solutions, suspensions, emulsions, and the like, pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or component by an appropriate method in the art.

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically applied) according to a desired method, and the dosage may be the patient's weight, age, sex, The range varies according to health status, diet, administration time, administration method, excretion rate, and severity of disease. The daily dosage of the composition is about 0.0001-500 mg/kg, preferably about 0.001-300 mg/kg, and is preferably administered once to several times a day.

The pharmaceutical composition of the present invention may be used alone or in combination with surgery, hormone therapy, drug therapy, and methods of using a biological response modifier for anti-inflammatory effect.

In the present invention, the term "health functional food" refers to a food having body control functions such as prevention and improvement of diseases, body defense, immunity, recovery after illness, and suppression of aging, and should be harmless to the human body when taken for a long time.

The composition of the present invention may be added to health functional foods for the purpose of improving inflammation. When the dogwood extract of the present invention is used as a food additive, the dogwood extract may be added as it is or used with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, when preparing food or beverage, the dogwood extract of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, the amount may be below the above range, and there is no problem in terms of safety, so the active ingredient may be used in an amount above the above range.

There is no particular limitation on the type of food. Examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, There are alcoholic beverages and vitamin complexes, and all health functional foods in the usual sense are included.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acids and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, Carbonating agents used in carbonated beverages may be contained. In addition, the composition of the present invention may contain flesh for the production of natural fruit juice, fruit juice beverage and vegetable beverage. These components may be used independently or in combination. The proportion of these additives is not very important, but it is generally selected in the range of 0.01-0.20 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, the present invention provides a method for treating inflammation comprising administering an anti-inflammatory pharmaceutical composition containing a fermented dogwood fermented extract as an active ingredient to an individual in need of treatment for inflammation.

The subject is a human or a mammal, including non-humans, and non-human mammals include, but are not limited to, mice, rats, dogs, cats, horses, cows, sheep, goats, pigs, rabbits, and the like.

Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. Experimental preparation and method

1.1. Material preparation

Dogwood used in this example ( Caryopterid incana ) was purchased commercially, dried in a 50°C dry oven (Jeiotech, Daejeon, Korea), pulverized with 40 mesh, separated into regular particles, vacuum-packed, and stored at a low temperature at 4°C and used as a sample.

1.2. Lactic acid fermentation production of dogwood

1.2.1. Lactic acid bacteria culture

The lactic acid bacteria ( Lactobacillus plantarum ) used in this example was a fungus isolated from the flounder sea, and was provided in the Food and Applied Microbiology Laboratory of the Department of Food Science and Technology, Kyungpook National University. For preservation of the strain, the bacterial culture was prepared to be 12% glycerol (Junsei Chemical, Tokyo, Japan) and stored in a -70°C quick freezer. After passage 1 in 5 mL of MRS broth (Difco ^TM Lactobacilli MRS, Becton Dickinson, USA) medium in a conical tube, inoculate the lactic acid bacteria culture solution in 300 mL of MRS broth medium in an Erlenmeyer flask, and then increase the culture in a 37°C incubator for 24 hours. I did. In order to measure the number of viable bacteria, 2 passages of lactic acid bacteria culture medium were diluted from 10 ^-1 to 10 ^-10 using MRS broth, and 0.1 mL was plated on MRS agar plate medium, and the average value was calculated.

1.2.2. Solid fermentation of dogwood

In this example, dogwood fermentation was fermented using a solid fermentation technique, that is, 5 mL of Lactobacillus plantarum culture solution was inoculated in a sterile condition to 10 g of a sterilized dogwood outpost dry powder sample, and then in a 37°C incubator for 24 hours. It was cultivated still, and it was periodically shaken every 6 hours to ensure even fermentation.

1.3 Preparation of dogwood extract

In the present example, 80% ethanol, which is the optimum condition for extraction using dogwood outpost, was selected as the extraction solvent to prepare non-fermented dogwood extract and fermented dogwood extract. 10 g of each of the non-fermented dogwood and fermented dogwood outpost powder was prepared, immersed in 100 mL of 80% ethanol, and extracted with stirring at room temperature for 24 hours, and the extract was made with Whatman No.1 filter paper (Whatman, Maidstone, UK). After filtration, all 80% ethanol, an extraction solvent, was removed and used using a rotary vacuum evaporator (Eyela NE, Tokyo, Japan). In order to measure the inhibitory effect of inflammation in Raw 264.7 cells, the filtered extract was lyophilized and stored at -20°C, and 100 mg was dissolved in 1 mL of dimethyl sulfoxide (DMSO) and used as a stock.

1.4. Determination of total phenolic component

In this example, the total phenolics content of each extract was measured by the Folin-Denis method, and 1 mL of 95% ethanol and 5 mL of distilled water were added to 1 mL of the sample extract, and 0.5 mL of 1 N Folin-ciocalteu reagent. Was mixed well, left for 5 minutes, and 1 mL of 5% Na ₂ CO ₃ was added. Absorbance was measured at 725 nm within 1 hour with a UV-visible spectrophotometer (Optizen 3220UV Mecasys, Daejeon, Korea), and the amount was converted from a standard curve using gallic acid.

1.5. Measurement of inhibitory effect on inflammation in Raw 264.7 cell line

1.5.1. Reagents and instruments

Reagents used for the anti-inflammatory efficacy assay were lipopolysaccharide (LPS), 3(4,5dime thylthiazol2yl)2,5diphenyltetrazolium (MTT) and DMSO were purchased from Sigma (St. Louis, MO, USA), and fetal bovine serum ( FBS) and streptomycin penicillin were purchased and used from Hyclone (Logan, UT, USA), and the primary antibodies GAPDH (Thermo, Wilmington, USA), COX2 (Cayman, Ann Arbor, USA), iNOS and the secondary antibody antirabbit IgG horseradish peroxidase (HRP) conjugated antibody and antimouse IgG1 horseradish peroxidase (HRP) conjugated antibody (Santa Cruz, CA, USA) were purchased respectively. For other reagents, special reagents were used, centrifuge (Hanil, Gimpo, Korea), dual minigel vertical unit and wet tank transfer (Hoefer, Holliston, MA, USA), electrophoresis power supply (Amersham pharmacia biotechnology, Piscataway, NJ) , SPECTRO star Nano ELISA reader (BMG LabTech, Ortenberg, Germany), C 300 image analyzer (Azure biosystems, Dublin, USA), etc. were measured using.

1.5.2. Cell culture

Raw 264.7 cells, a murine macrophage cell line, were purchased from the Korean Cell Line Research Foundation, and Hyclone fetal bovine serum (FBS) was added 10%, 100 U/mL penicillin, and 100 in Dulbecco's modified Eagle's medium (DMEM). Incubated for 48 hours in a 5% CO ₂ incubator at 37°C using a medium mixed with μg/mL streptomycin.

1.5.3. Cytotoxicity measurement by MTT assay

Cytotoxicity was measured by MTT assay. Raw 264.7 cells were cultured in a 48 well plate with 1×10 ⁴ cells in a 37°C incubator supplied with 5% CO ₂ for 24 hours, and then replaced with a serum-free medium. After adding 0.05 mL of the sample prepared by concentration, it was incubated for 24 hours in a 37°C, 5% CO ₂ incubator. Add 0.05 mL of MTT solution prepared at a concentration of 3 mg/mL to incubate for 4 hours, remove the culture medium, add 0.5 mL of DMSO to each well and react at room temperature for 10 minutes, and then enzyme-linked immunosorbent assay (ELISA) reader Absorbance was measured at 540 nm. Cytotoxicity was measured by the rate of decrease in absorbance of the sample solution added group and non-added group. The control group was cultured under the same conditions by adding the same amount of serum-free medium as the sample.

1.5.4. Measurement of iNOS and COX 2 protein expression through Western blot

After preparing a sample, 5×10 ⁵ cells were cultured in a 37°C incubator supplied with 5% CO ₂ for 24 hours in a 6 well plate, and then replaced with a serum-free medium. 10 μg/mL of LPS was added to all wells except for the normal group to stimulate, and the sample group was treated with samples prepared by concentration and incubated for 24 hours, and the supernatant was removed at an appropriate time and washed with cold PBS. 40 μL of lysis buffer mixed with Mammalian protein extraction reagent (MPER) and protease inhibitor cocktail (ThermoFischer Sxientific, San Jose, CA, USA) was added to destroy the cell membrane, followed by centrifugation at 4°C and 13,000 rpm for 15 minutes. The supernatant was collected and transferred to a new tube, and then the amount of protein was corrected by substituting the OD value into a standard curve prepared with BSA (bovine serum albumin). After 25 μL of protein was separated by electrophoresis using 10% SDSpolyacrylamide gel, the gel was removed and the PVDF membrane and gel were well adhered between both sponges and 3mm paper, and then transferred at 60 V for 2 hours and 30 minutes. . The background was removed by blocking with 5% skim milk solution at room temperature for 1 hour. The primary antibody was diluted 1:200, over night at 4°C, and washed 3 times with 1X TBST. The secondary antibody was diluted 1:200 for iNOS and 1:1000 for COX2, attached at room temperature for 1 hour 30 minutes, and washed 3 times with 1X TBST. It reacted with an ECL kit (EZ west Lumi plus, ATTO, Tokyo, Japan) and exposed to an X-ray film, followed by band development and quantification using a C 300 image analyzer (Azure biosystems, Dublin, USA).

1.5.5. Nitric oxide (NO) 　 measurement

NO amount of nitrite present in a secure form to the supernatant (NO ₂ ^-) a reduction, which was measured using a griess reagent (Promega, USA) reaction. 5 × 10 ⁴ cells in a 96 well plate were incubated for 24 hours in a 37°C incubator supplied with 5% CO ₂ , and then replaced with a serum-free medium. 10 μg/mL of LPS was added to all wells except the normal group and stimulated. The sample group was treated with samples prepared by concentration, incubated for 24 hours, collected supernatant, and reacted with griess reagent, and absorbance at 540 nm. Measured.

1.5.6. Prostaglandin E ₂ (PGE ₂ ) Measurement of expression inhibition effect

After incubating 1×10 ⁵ cells in a 96 well plate in a 37°C incubator supplied with 5% CO ₂ for 24 hours, they were exchanged for serum-free medium. LPS 2.5 μg/mL was added to all wells except the normal group and stimulated. The sample group was treated with samples prepared by concentration and incubated for 24 hours, and then the supernatant was taken and used to measure the effect of inhibiting PGE ₂ expression. PGE ₂ Measurement was performed according to the experimental method of ELISA kits (R&D Systems. USA & Canada). Calibrator diluent RD5-56 was added 200 μL to non specific binding (NSB) wells, 150 μL to zero standard (B ₀ ) wells, and 150 μL of standard, control, and sample to the remaining wells. 50 μL of primary antibody solution was added to each well excluding NSB wells, covered with a plate cover, and shaken for 1 hour at room temperature, followed by washing 4 times with a PGE ₂ washing buffer. After washing, 200 μL of the substrate solution was added, and the reaction was performed for 30 minutes by blocking light. After the reaction, 100 μL of the stop solution was added and the absorbance was measured at 450 nm, and the amount was converted into a standard curve using a standard.

1.5.7. Measurement of cytokine production

1.5.7.1. Measurement of Tumor necrosis factorα (TNFα) inhibitory activity

After incubating 3×10 ⁵ cells in a 96 well plate in a 37°C incubator supplied with 5% CO ₂ for 24 hours, they were replaced with serum-free medium. LPS 2.5 μg/mL was added to all wells except for the normal group to stimulate, and the sample group treated samples prepared by concentration and cultured for 24 hours, and a supernatant was taken and used for TNFα measurement. TNFα was measured according to the experimental method of ELISA kit (R&D Systems. USA & Canada). After 50 μL of sample and standard were added to the well plate, 50 μL of biotinylated antibody reagent was added each. The plate cover was covered and incubated for 2 hours at room temperature, followed by washing 5 times using a TNFα washing buffer. 100 μL of StreptavidinHRP solution was added and incubated at room temperature for 30 minutes. After washing 5 times using a TNFα washing buffer, 100 μL of TMB substrate solution was added. After blocking the light and incubating for 30 minutes, adding 100 μL of stop solution each to terminate the reaction, the absorbance was measured at 450 nm, and the amount was converted into a standard curve using a standard.

1.5.7.2. Measurement of interleukin6 (IL6) inhibitory activity

After incubating 1×10 ⁵ cells in a 96 well plate in a 37°C incubator supplied with 5% CO ₂ for 24 hours, they were replaced with serum-free medium. LPS 2.5 μg/mL was added to all wells except for the normal group to stimulate, and the sample group treated samples prepared by concentration and incubated for 24 hours, and then a supernatant was taken and used for IL6 measurement. IL-6 Measurement was performed according to the experimental method of ELISA kits (R&D Systems. USA & Canada). After 50 μL of assay diluent RD1-14 was added to a 96 well plate, 50 μL of standard, control, and sample were added. The well plate cover was covered and shaken for 2 hours, and then washed 5 times using IL6 washing buffer. After adding 100 μL of IL6 conjugate, the well plate cover was covered and shaken for 2 hours. After washing 5 times using an IL6 washing buffer, 100 μL of a substrate solution was added, and the light was blocked, followed by shaking for 30 minutes. The reaction was terminated by adding 100 μL of Stop solution, and absorbance was measured at 450 nm, and the amount was converted to a standard curve using standard.

1.5.7.3. Measurement of Interleukin-1β (IL1β) inhibitory activity

After incubating 1×10 ⁵ cells in a 96 well plate in a 37°C incubator supplied with 5% CO ₂ for 24 hours, they were exchanged for serum-free medium. LPS 2.5 μg/mL was added to all wells except the normal group for stimulation, and the sample group treated samples prepared by concentration and incubated for 24 hours, and then the supernatant was taken and IL1β was used for measurement. IL1β measurement was performed by ELISA kits R&D Systems. USA & Canada) was measured according to the experimental method. After 50 μL of assay diluent RD1-14 was added to a 96 well plate, 50 μL of standard, control, and sample were added. The well plate cover was covered and shaken for 2 hours, and then washed 5 times using IL1β washing buffer. After adding 100 μL of IL1β conjugate, the well plate cover was covered and shaken for 2 hours. After washing 5 times with an IL1β washing buffer, 100 μL of a substrate solution was added, and the light was blocked and shaking for 30 minutes. The reaction was terminated by adding 100 μL of Stop solution, and absorbance was measured at 450 nm, and the amount was converted to a standard curve using standard.

Example 2. Comparison of elution amount of phenolic compound according to solvent type and concentration change of dogwood extract

In order to establish the extraction conditions of the dogwood extract, the elution amount of the phenolic compound expected to be involved in physiological activity from the dogwood tree was confirmed according to the method of Example 1.4. by varying the extraction solvent and concentration.

As a result, as shown in Figure 1, when extracted with ethanol (ethanol), 11.47 mg / g, the elution amount of the phenolic compound was the highest among the solvents used, followed by methanol (methanol), butanol (butanol) ), water, and acetone in order. Thus, for efficient extraction of phenolic compounds, the ethanol concentration was set and extracted step by step.

As a result, as shown in Fig. 2, the elution amount of the phenolic compound was highest in 80%　ethanol extract at 12.50 mg/g. According to the above results, the anti-inflammatory efficacy of the non-fermented product and the fermented product of 80% ethanol extract of Japanese dogwood were compared.

Example 3. Comparison of anti-inflammatory effects of non-fermented ethanol extract and fermented ethanol extract of dogwood

3.1. Cytotoxicity check

In order to confirm the cytotoxicity of non-fermented dogwood ethanol extract and fermented ethanol extract in Raw 264.7 cells, non-fermented dogwood ethanol extract and fermented ethanol extract were used by concentration (5, 10, 15, 20, 30, 40, 50 µg/mL), and then the MTT assay was performed according to the method of Example 1.5.3. to measure cytotoxicity.

As a result, as shown in Figs. 3a and 3b, both the non-fermented dogwood ethanol extract (see Fig. 3a) and fermented ethanol extract (see Fig. 3b) tend to significantly decrease the cell viability as the concentration of the extract increases. Shown. In particular, cell viability decreased by 79.27% in the group treated with 20 µg/mL of non-fermented dogwood ethanol extract, resulting in cytotoxicity. In the case of the fermented dogwood ethanol extract, cell viability was 77.7% in the 50 µg/mL treatment group. It was observed that the decrease was observed, and it was confirmed that the toxicity of the extract was lowered after fermentation.

Therefore, in the case of the non-fermented dogwood ethanol extract, the experiments were conducted at concentrations of 5, 10, and 15 µg/mL, which did not affect the viability of the cells, and the fermented dogwood ethanol extract did not affect the survival rate of cells. Subsequent experiments were performed at concentrations of 10, 20, 30, and 40 ㎍/mL.

3.2. INOS and COX via Western Blot 2 Confirmation of protein expression inhibitory effect

In order to confirm the effect of inhibiting the expression of inducible nitric oxide synthase (iNOS) protein of the non-fermented dogwood ethanol extract and the fermented ethanol extract, iNOS was performed by performing western blot according to the method of Example 1.5.4. The amount of protein expression was measured.

As a result, as shown in FIGS. 4a and 4b, according to the band density ratio of GAPDH, the non-fermented dogwood ethanol extract (see FIG. 4a) and fermented dogwood ethanol extract (see FIG. 4b) were respectively 15 μg/mL and At a concentration of 40 μg/mL, it exhibited an effect of inhibiting expression of about 50%. From the above, it was confirmed that the effect of inhibiting iNOS expression of the non-fermented dogwood ethanol extract and the fermented dogwood ethanol extract was very excellent.

In addition, in order to confirm the effect of inhibiting the expression of Cyclooxygenase2 (COX2) protein of non-fermented dogwood ethanol extract and fermented ethanol extract during lipopolysaccharide (LPS) treatment, according to the method of Example 1.5.4. Blot was performed.

As a result, as shown in FIGS. 5A and 5B, it was confirmed that the amount of COX2 protein expression decreased in a concentration-dependent manner. According to the band density ratio of GAPDH, the non-fermented dogwood ethanol extract was 50% at a concentration of 15 μg/mL. (See Fig. 5a), the fermented dogwood ethanol extract exhibited an expression inhibitory effect of 17% at a concentration of 40 µg/mL (see Fig. 5b). From the above, it was determined that during the inflammatory reaction, the non-fermented dogwood ethanol extract and the fermented dogwood ethanol extract reduced the expression level of COX2 protein to show anti-inflammatory effect, and thus could be used as a useful resource for the development of immunity enhancing materials.

3.3. Confirmation of inhibitory effect on the formation of nitric oxide (NO)

In order to measure the degree of inhibition of NO production in raw 264.7 cells, the amount of NO produced by treating cells with non-fermented dogwood ethanol extract and fermented dogwood ethanol extract at various concentrations was measured according to the method of Example 1.5.5. I did.

As a result, as shown in FIGS. 6A and 6B, the Normal (NOR) group showed nearly five times more NO generation than the control (CON) group, and the non-fermented dogwood ethanol extract inhibited NO generation as the concentration increased. The effect was observed to be high, and there was an effect of inhibiting NO generation of 40% at a concentration of 15 μg/mL (see FIG. 6A). Fermented dogwood ethanol extract exhibited a similar level of inhibition of NO generation as in the normal group at a concentration of 40 μg/mL (see FIG. 6B). From the above, it was confirmed that the non-fermented dogwood ethanol extract and the fermented dogwood ethanol extract had excellent NO production inhibitory effect.

3.4. Prostaglandin E ₂ (PGE ₂ ) Checking the effect of inhibiting expression

To examine the effect of the non-fermented dogwood ethanol extract and the fermented dogwood ethanol extract on the expression of PGE ₂ produced by LPS in Raw 264.7 cells, the amount of PGE ₂ produced was determined according to the method of Example 1.5.6. Measured.

As a result, as shown in FIGS. 7a and 7b, PGE ₂ was about 15 μg/mL in non-fermented dogwood ethanol extract (see FIG. 7a), and about 30 μg/mL in fermented dogwood ethanol extract. It showed a 30% expression inhibition rate (see Fig. 7b), A concentration-dependent expression inhibition phenomenon could be confirmed. From the above, it was determined that the effect of inhibiting the high inflammatory response in Raw 264.7 cells, a macrophage cell line induced by LPS, such as the suppression of the expression of the COX2 protein can be expected.

Example 4. Comparison of Cytokine Inhibitory Activity of Non-fermented Ethanol Extract and Fermented Ethanol Extract of Dogwood

4.1. Confirmation of Tumor necrosis factorα (TNFα) inhibitory activity

To examine the effect of non-fermented dogwood ethanol extract and fermented dogwood ethanol extract on the activity of TNFα, which plays a major role in various inflammatory diseases such as rheumatoid arthritis and septic shock, according to the method of Example 1.5.7.1, above. The expression level of TNFα was measured.

As a result, as shown in Figs. 8a and 8b, TNFα exhibited an expression inhibitory effect of about 60% at a concentration of 15 µg/mL in the non-fermented dogwood ethanol extract (see FIG. 8a), and the fermented dogwood ethanol extract Showed an expression inhibitory effect of about 35% at a concentration of 40 μg/mL (see FIG. 8B).

4.2. Confirmation of interleukin6 (IL6) inhibitory activity

In order to examine the effect of the non-fermented dogwood ethanol extract and the fermented dogwood ethanol extract on the activity of IL6 synthesized in various types of cells as a multidirectional cytokine, the expression level of IL6 was determined according to the method of Example 1.5.7.2. Measured.

As a result, as shown in Figs. 9a and 9b, IL6 is about 35 at a concentration of 15 µg/mL for non-fermented dogwood ethanol extract (see Fig. 9a), and about 35 for fermented dogwood ethanol extract at a concentration of 40 µg/mL. % Of the expression inhibitory effect was shown (see Fig. 9b).

4.3. Confirmation of interleukin1β (IL1β) inhibitory activity

In order to examine the effect of the non-fermented dogwood ethanol extract and the fermented dogwood ethanol extract on the activity of IL1β, which induces an inflammatory reaction and is known as a heat-resistant substance and lymphocyte activating factor, according to the method of Example 1.5. The expression level of IL1β was measured.

As a result, as shown in FIGS. 10A and 10B, IL1β showed an expression inhibitory effect of about 48% at a concentration of 15 μg/mL in the non-fermented dogwood ethanol extract (see FIG. 10A), and the fermented dogwood ethanol extract Showed an expression inhibitory effect of about 30% at a concentration of 40 μg/mL (see FIG. 10B).

The above-described description of the present invention is for illustration purposes only, and those of ordinary skill in the art to which the present invention pertains can understand that it is possible to easily transform it into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not limiting.

Claims (17)

An anti-inflammatory pharmaceutical composition containing solid fermented dogwood fermented extract with lactic acid bacteria ( Lactobacillus plantarum ) as an active ingredient,
The extract is 80% ethanol extract, characterized in that, anti-inflammatory pharmaceutical composition.
delete delete The method of claim 1,
The solid fermentation extract is characterized in that it exhibits an effect of inhibiting the expression of inducible nitrogen oxide synthase (iNOS), anti-inflammatory pharmaceutical composition.
The method of claim 1,
The solid fermentation extract is characterized in that it exhibits the effect of inhibiting the expression of cyclooxygenase (COX2), anti-inflammatory pharmaceutical composition.
The method of claim 1,
The solid fermentation extract is characterized in that it exhibits a nitrogen oxide (NO) production inhibitory effect, anti-inflammatory pharmaceutical composition.
The method of claim 1,
The solid fermentation extract is characterized in that it exhibits an inhibitory effect on prostaglandin E ₂ (PGE ₂ ) production, anti-inflammatory pharmaceutical composition.
The method of claim 1,
The solid fermented extract shows an effect of inhibiting the expression of at least one cytokine selected from the group consisting of tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and interleukin-1β (IL-1β). Characterized in, anti-inflammatory pharmaceutical composition.
It is a health functional food composition for improving inflammation containing solid fermented dogwood fermented extract from solid fermentation with lactic acid bacteria ( Lactobacillus plantarum ) as an active ingredient,
The extract is a health functional food composition for improving inflammation, characterized in that 80% ethanol extract.
delete delete The method of claim 9,
The solid fermentation extract is characterized in that it exhibits an effect of inhibiting the expression of inducible nitrogen oxide synthase (iNOS), a health functional food composition for improving inflammation.
The method of claim 9,
The solid fermentation extract is characterized in that it exhibits the effect of inhibiting the expression of cyclooxygenase (COX2), health functional food composition for improving inflammation.
The method of claim 9,
The solid fermentation extract is characterized in that it exhibits a nitrogen oxide (NO) production inhibitory effect, health functional food composition for improving inflammation.
The method of claim 9,
The solid fermented extract is a health functional food composition for improving inflammation, characterized in that it exhibits an inhibitory effect on prostaglandin E ₂ (PGE ₂ ) production.
The method of claim 9,
The solid fermented extract shows an effect of inhibiting the expression of at least one cytokine selected from the group consisting of tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and interleukin-1β (IL-1β). Characterized in, a health functional food composition for improving inflammation.
a) solid fermentation by inoculating the Lactic Acid Bacteria culture solution on the dogwood tree; And
b) extracting the solid fermented dogwood by immersion in 80% ethanol;
A method for preparing the composition of claim 1 comprising a.

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