KR102587456B1 - Anti―inflammatory pain composition using bacteria fermentation-microwave extract of arnica angustifolia - Google Patents

Abstract

The present invention discloses an anti-inflammatory analgesic composition using a fermented extract of Arnica Montana. The fermented extract is an ultrasonic extract using Arnica Montana and Bhuiamala , Enterococcus faecium , Lactobacillus plantarum , Bifidobacterium longum , Lactobacillus acidophilus ( Lactobacillus acidophilus ), and It is obtained by inoculating one or more fermentation strains selected from Leuconostoc mesenteroides , and is extracted with alcohol to provide anti-inflammatory and analgesic effects.

Description

ANTI-INFLAMMATORY PAIN COMPOSITION USING BACTERIA FERMENTATION-MICROWAVE EXTRACT OF ARNICA ANGUSTIFOLIA}

The present invention relates to an anti-inflammatory analgesic composition capable of treating sprains, bruises, muscle pain, joint pain, fracture pain, back pain, stiff shoulders, neuralgia, rheumatic pain, skin itchiness, insect bites, and chilblains using a fermented extract of Arnica. will be.

Arnica Montana ( Arnica Montana ) is a flower belonging to the Aster family of the Asteraceae family. It grows in the grasslands of the highlands and is 20 to 30 cm tall. Leaves emerge from the roots, spread in all directions, and run opposite each other on the stem. The branches split at the end and one to three heads grow. The flowers bloom in yellow from June to July, are 6 to 8 cm in diameter, and are densely covered with glandular hairs and hairs. The flowers and rhizomes have a bitter taste and contain essential oil and resin. The European folk have used flowers and rhizomes as an all-purpose medicine since ancient times. In oriental medicine, it is also used as a hemostatic agent for stimulating angina, dilating blood vessels, alleviating vascular spasm, and treating bruises and hemorrhoids. Arnica montana of the present invention uses whole plants among various organs or parts (e.g. leaves, bark, branches, flowers, roots, fruits, stems, etc.).

Bhui amala ( Bhui amala ) is a tropical plant with the scientific name Phyllanthus niruri, and is a soft, light green herbaceous plant. It has been used in traditional medicine for a variety of ailments, including jaundice, chronic dysentery, indigestion, cough, diabetes, urinary tract disorders, skin diseases, ulcers, and swelling. Phyllanthus niruri can be harvested and used as the whole plant, or optionally one or more parts of the plant (e.g., flowers, seeds, roots, rhizomes, stems, fruits and/or leaves of the plant) can be used. The Phyllanthus niruri plant or parts thereof may be ground into a powder, for example by grinding or milling. However, nothing has been disclosed or disclosed anywhere in the literature regarding the anti-inflammatory analgesic performance of V Amala.

Accordingly, while conducting research on Arnica and V Amala, the present inventors completed the present invention by confirming that they can exhibit anti-inflammatory analgesic properties, including sprains and bruises.

Korean Patent Publication No. 2016-0070947

The purpose of the present invention is to simultaneously express sprains, bruises, muscle pain, joint pain, fracture pain, back pain, stiff shoulders, neuralgia, rheumatic pain, skin itchiness, insect bites, and chilblains using a fermented extract of Arnica Montana. The aim is to provide a composition that does.

The above and other objects of the present invention can all be achieved by the present invention described below.

In order to achieve the above object, the present invention

Enterococcus faecium , Lactobacillus plantarum , Bifidobacterium longum , and Lactobacillus acido were added to the ultrasonic extract using Arnica Montana and Bhuiamala . Pilus ( Lactobacillus acidophilus ), and An anti-inflammatory and analgesic composition comprising a powder of a fermented product obtained by inoculating one or more fermentation strains selected from Leuconostoc mesenteroides or an extract thereof as an active ingredient is provided.

The powder or extract can be obtained by alcohol extraction of the fermented product.

The alcohol extraction may be performed under reflux at 60 to 70°C for 2 to 3 hours.

The ultrasonic extract can be extracted for 10 to 20 minutes at a frequency of 2200 to 2500 MHz.

The fermentation strain may be Enterococcus faecium (KCTC 13225) or Lactobacillus plantarum (KCCM 11322) .

The anti-inflammatory analgesic may be used to treat sprains, bruises, muscle pain, joint pain, fracture pain, back pain, stiff shoulders, neuralgia, rheumatic pain, skin itchiness, insect bites, or chilblains.

According to the present invention, fermentation of arnica is capable of relieving pain through anti-inflammatory analgesic properties in sprains, bruises, muscle pain, joint pain, fracture pain, back pain, stiff shoulders, neuralgia, rheumatic pain, itchy skin, insect bites, or chilblains. A powder of the extract or an anti-inflammatory analgesic composition containing the extract as an active ingredient can be provided.

Figure 1 is a graph comparing cell viability evaluation results for the sample obtained in Example 1 and the four samples obtained in Comparative Examples 1 to 3.
Figure 2 is a graph comparing NO concentration evaluation results for the sample obtained in Example 1 and the four samples obtained in Comparative Examples 1 to 3.
Figure 3 is a graph quantifying the results of cytokine concentration evaluation for the sample obtained in Example 1 and the four samples obtained in Comparative Examples 1 to 3.
Figure 4 is a graph comparing the arthritis severity evaluation results in mice with collagen-induced arthritis using the sample obtained in Example 1 and the four samples obtained in Comparative Examples 1 to 3.
Figure 5 is a graph comparing cytokine concentrations in mice with collagen-induced arthritis using the sample obtained in Example 1 and the four samples obtained in Comparative Examples 1 to 3.

Hereinafter, the present invention will be described in more detail to facilitate understanding of the present invention.

Terms and words used in this specification and claims should not be construed as limited to their common or dictionary meanings, and the inventor may appropriately define the concept of terms in order to explain the invention in the best way. Therefore, it should be interpreted with meaning and concept consistent with the technical idea of the present invention.

As confirmed in the examples and experimental examples below, the present invention includes Enterococcus faecium and Lactobacillus plantarum in ultrasonic extracts using Arnica Montana and Bhuiamala . ), Bifidobacterium longum , Lactobacillus acidophilus , and When producing a fermented product obtained by inoculating one or more fermentation strains selected from Leuconostoc mesenteroides , the powder or its extract may cause sprains, bruises, muscle pain, joint pain, fracture pain, back pain, stiff shoulders, and neuralgia. , was completed by confirming that it has the ability to treat rheumatic pain, skin itchiness, insect bites, or chilblains.

Unfermented Arnica extract did not show sufficient anti-inflammatory and analgesic effect not only for ethanol extract and Arnica ultrasonic extract, but also for extracts subjected to both ultrasonic and ethanol extraction. Additionally, although not presented in the experimental example below, Arnica Montana ( Arnica Montana ) and Bhuiamala on solid dried materials such as Enterococcus faecium, Lactobacillus plantarum, Bifidobacterium longum , and Lactobacillus acidophilus. acidophilus ), and The powder of the fermented product obtained by inoculating one or more fermentation strains selected from Leuconostoc mesenteroides or its extract did not show sufficient muscle function enhancement effect.

The composition that implements anti-inflammatory analgesia using arthritis and cytokine concentration in the cell viability, NO concentration, cytokine concentration, and collagen-induced arthritis animal model of the present invention is provided based on these experimental results. The anti-inflammatory analgesic composition of the present invention is Arnica Montana and Bhuiamala were mixed at a weight ratio of 1:0.5 to 1.5, then extracted ultrasonically, and fermented bacteria such as Enterococcus faecium , Lactobacillus plantarum , and Bifidobacterium were added. Bifidobacterium longum , Lactobacillus acidophilus , and It is characterized in that it contains as an active ingredient a powder of a fermented product obtained by inoculating one or more types of lactic acid bacteria selected from Leuconostoc mesenteroides or an extract thereof.

In this description, unless otherwise specified, an active ingredient refers to an ingredient that exhibits the desired activity alone or can exhibit activity in combination with a carrier that is not active on its own.

The ultrasonic treatment is a treatment method that uses energy generated by ultrasonic vibration. Ultrasonic waves can destroy the insoluble solvent contained in the sample in an aqueous solvent, and the high local temperature generated at this time causes the surrounding reactant particles to be destroyed. By increasing the kinetic energy, sufficient energy required for the reaction is obtained, and the shock effect of ultrasonic energy induces high pressure to increase the mixing effect of the substances contained in the sample and the solvent, thereby increasing extraction efficiency.

Extraction solvents that can be used in the ultrasonic extraction method include, for example, one or a mixture of two or more selected from chloroform, ethanol, methanol, water, ethyl acetate, nucleic acid, and diethyl ether.

As shown in the examples described later, it is preferable to use alcohol as a solvent during ultrasonic treatment because it provides improved anti-inflammatory analgesic performance when alcohol is extracted after ultrasonic treatment.

The ultrasonic extraction can be performed for 10 to 20 minutes at a frequency of 2200 to 2500 MHz. In this case, it can provide increased extraction efficiency of effective substances.

It can be obtained by alcohol extraction of the fermented product of the ultrasonic extract.

The ultrasonic extract includes Enterococcus faecium , Lactobacillus plantarum , Bifidobacterium longum , Lactobacillus acidophilus , and One or more types of lactic acid bacteria selected from Leuconostoc mesenteroides are added at 10 ⁴ to 10 ⁷ CFU/g, for example, 10 ⁵ to 10 ⁶ CFU/g.

In the present invention, the fermentation strain is preferably Enterococcus faecium (KCTC 13225) or Lactobacillus plantarum (KCCM 11322) used in the examples below to provide an anti-inflammatory and analgesic composition, and in addition, Bifidobacterium longum . , Lactobacillus acidophilus, Leuconostoc mesenteroides , etc. can also be used as replacements or combinations.

In the present invention, the fermentation bacteria are lactic acid bacteria activated with Arnica Montana extract prior to inoculation, specifically those obtained by inoculating and culturing the lactic acid bacteria on a medium containing Arnica water (including hot water) extract and carbon sources such as sugars. It may be possible. Although not presented in the experimental example below, the present inventors believe that when lactic acid bacteria activated with arnica water (including hot water) extract are used, fermentation will proceed to the core of the ultrasonic extract (pulverized material). Lactic acid bacteria activated with this Arnica water (including hot water) extract can be obtained by preparing a medium containing the Arnica water extract and a carbon source and inoculating it with lactic acid bacteria, where the carbon source is any carbon source known in the art, such as Lifting oligosaccharides, lactose, glucose, fructose, sucrose, sugar, dextrose, starch, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose and mixtures thereof can be used. The amount added to the medium may be in the range of 1 to 10% (w/v), and the medium may further contain a nitrogen source or trace element in the range of 0.1 to 3% (w/v), with ammonia as the nitrogen source. , ammonium salts, urea, amino acids, soybean meal, soybean protein, yeast extract, meat juice, etc., and trace elements include K, Mg, Na, Co, Mo, Ca, Mn, Zn, Cu, Fe, P, S, etc. can be mentioned. Additionally, culturing may be performed at 25 to 40°C or 35 to 37°C for about 12 hours to 2 days.

In the present invention, the culture after inoculation may be performed at 15 to 45°C, 30 to 40°C, or 35 to 37°C. If the culture temperature is lower than the above range, the fermentation speed may be slowed or unwanted by-products may be generated. Even if the culture temperature is higher than the above range, the fermentation speed may be slowed or unwanted fermentation by-products may be formed due to the death of fermentation microorganisms. . Cultivation time may be 1 to 7 days, or 2 to 5 days.

Then, the supernatant is extracted by centrifugation according to any known method, and the extract can be extracted from the supernatant thus obtained.

At this time, unless otherwise specified, the extract of the above-mentioned supernatant (i.e., the supernatant of the fermented product) is extracted by mixing the fermented product supernatant itself, which is the object of extraction, with water, lower alcohols with 1 to 4 carbon atoms (methanol, ethanol, butanol, etc.), methylene chloride, acetone, Supercritical extraction of extracts obtained by leaching using hexane, ether, chloroform, ethyl acetate, butylacetate, dimethylformamide, dimethyl sulfoxide, 1,3-butylene glycol, propylene glycol or mixed solvents thereof, carbon dioxide, pentane, etc. It refers to an extract obtained using a solvent or a fraction obtained by fractionating the extract, and the extraction method is any method such as cold immersion, reflux, heating, ultrasonic radiation, or supercritical extraction, considering the polarity of the active substance, degree of extraction, and degree of preservation. can be applied.

In the present disclosure, extraction at high temperature with a lower alcohol having 1 to 4 carbon atoms is appropriate to provide optimal extraction conditions for the active ingredient. Preferably, it can be performed under reflux. At this time, the high temperature may be 85°C or lower, for example, 50 to 80°C, and the extraction time is not limited thereto, but extraction may be repeated for 12 hours or less, for a specific example, 1 to 6 hours. The number of repeated extractions may be 5 times or less, for example, 2 to 3 times.

In addition, it is preferable to perform reflux extraction to return the water vapor into the reactor and discharge the remaining gases to maximize extraction efficiency.

In the case of fractionated extracts, the extract is suspended in a specific solvent, mixed with a solvent of different polarity, and the obtained fractions and crude extract are adsorbed on a column filled with silica gel, etc., and then a hydrophobic solvent, a hydrophilic solvent, or a mixed solvent thereof is used as the mobile phase. It is meant to include fractions obtained by.

The meaning of the extract includes concentrated liquid extract or solid extract from which the extraction solvent has been removed by methods such as freeze-drying, vacuum drying, hot air drying, spray drying, etc. Preferably, it refers to an extract obtained using water, a lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, butanol, etc.), or a mixed solvent thereof as an extraction solvent.

In the present disclosure, the form of the resulting extract is not particularly limited and may be in the form of pills, granules, powder, powder, or other extract forms.

In the present disclosure, Arnica Montana and Bhuiamala can be obtained in solid form after being thoroughly washed. After cleansing, some of the moisture is removed by drying in any method such as natural drying (shade drying) or hot air drying (in the form of pulverized material so that the moisture content of the solid is less than 10% (w/w)). It can be crushed to 50 mesh, specifically 30 to 50 mesh, and then used for lactic acid bacteria fermentation.

Furthermore, it can be used as is in the form of pulverized product or in a step-by-step steamed and then dried state, and it is preferable to use it in a step-by-step steamed and then dried state as it can provide an increased effect of the active ingredients.

The anti-inflammatory analgesic composition of the present invention may be prepared including a dispersant and a carrier in addition to the active ingredient.

The dispersant includes water, alcohol (e.g., methanol, ethanol, ethylene glycol, propylene glycol, diethylene glycol, glycerin, etc.), ketone (e.g., acetone, methyl ethyl ketone, etc.), and ether (e.g., Dioxane, tetrahydrofuran, cellosolve, diethylene glycol dimethyl ether, etc.), aliphatic hydrocarbons (e.g., hexane, kerosene, etc.), aromatic hydrocarbons (e.g., benzene, toluene, xylene, naphthalene, methylnaphthalene, etc.) ), halogenated hydrocarbons (e.g., chloroform, carbon tetrachloride, etc.), acid amides (e.g., dimethylformamide), esters (e.g., methyl acetate ester, ethyl acetate ester, butylacetate ester, fatty acid glycerin ester) etc.), nitriles (e.g. acetonitrile, etc.), surfactants (e.g. higher alcohol sulfate esters, alkyl sulfonic acids, alkyl allyl sulfonic acids, quaternary ammonium salts, oxyalkylamines, fatty acid esters, polyalkylene oxide compounds) , anhydrous sorbitol compounds, etc.), and may be prepared singly or in a mixture of two or more types included in the antibacterial composition of the present invention.

If necessary, the above-described dispersant may be applied to the fermented product and used to produce a capture body, a collection aggregate, or an extract thereof. Specifically, as in the example below, 60 to 70% ethanol, 10 to 20 times the weight by weight, can be added to the supernatant of the fermented product and extracted by connecting a reflux tube at 70°C for 3 hours. The obtained extract can be provided as an extract by filtering through a 0.45 ㎛ membrane filter and then concentrating in vacuum and room temperature for 3 hours.

For carriers, clay (e.g. kaolin, bentonite, acid viscosity, etc.), talc (e.g. talcum powder, pyrophyllite powder, etc.), silica (e.g. diatomaceous earth, silicic anhydride, mica powder, etc.), alumina, sulfur. Examples may include powder, activated carbon, etc., and these carriers may also be prepared individually or in a mixture of two or more types included in the anti-inflammatory analgesic composition of the present invention.

The composition of the present invention can be used in a variety of applications requiring anti-inflammatory pain relief. The specific effect or its use can be understood as the ability to treat sprains, bruises, muscle pain, joint pain, fracture pain, back pain, stiff shoulders, neuralgia, rheumatic pain, skin itching, insect bites, or chilblains.

Regarding the specific liquid formulation or formulation of the anti-inflammatory analgesic composition, various techniques known in the art can be applied.

When considered as the above-mentioned anti-inflammatory analgesic composition, the composition may take any product form, specifically solution, suspension, emulsion, paste, gel, cream, lotion, powder, cleansing, oil, powder. It can take the form of products such as powder, emulsion powder, wax powder, solvent, and lay.

In the case of the above-described anti-inflammatory analgesic composition, in addition to the active ingredient, commonly used ingredients such as solvents, stabilizers, solubilizers, emulsifying agents, diluents, suspending agents, surfactants, vitamins, colorants, and flavorings are added. May contain supplements.

For example, in the case of powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. can be used as the carrier, and especially in the case of spray, chlorofluorohydrocarbon, propane, butane or It may additionally contain a propellant such as dimethyl ether.

The solvent, solubilizing agent or emulsifying agent includes water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, fatty acid ester of sorbitan, etc. It can be used.

The diluent or suspending agent may be a liquid diluent such as water, ethanol, or propylene glycol, or a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, polyoxyethylene sorbitan ester, or microcrystalline cellulose or aluminum. Metahydroxide, bentonite, agar, etc. can be used.

The composition of the present invention may also be considered a pharmaceutical composition in specific embodiments. When the composition of the present invention is understood as a pharmaceutical composition, its pharmacological effect or its use can be understood as anti-inflammatory and analgesic treatment.

Furthermore, in addition to the above-mentioned aspects, it can be used without limitation for any application requiring the development of anti-inflammatory pain relief.

The anti-inflammatory analgesic composition and pharmaceutical composition of the present invention can be prepared according to the method for producing the composition commonly performed in the art, except that it contains active ingredients known in the art.

In describing the anti-inflammatory analgesic composition of the present disclosure, it is stated that other conditions or equipment not explicitly described can be appropriately selected within the range commonly practiced in the art and are not particularly limited.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily practice it. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

[Example]

<Example 1> Preparation of fermented arnica extract

Arnica Montana and Bhuiamala were purchased respectively.

(1) Arnica Montana and Bhuiamala were thoroughly washed, dried, and ground to a 40 mesh. The pulverized raw materials were mixed at a constant weight (100 g: 100 g).

(2) 70% ethanol, 10 times the weight by weight, was added to the dried raw material, and extraction was performed using a microwave extractor (Soxwave 100 product, stabilized after setting to 210W) at a frequency of 2,450 MHz for 15 minutes.

(3) Enterococcus faecium (KCTC 13225) was mixed and inoculated into the ultrasonic extract at a concentration of 10 ⁶ CFU/g and fermented at 30°C for 3 days.

(4) The culture medium was centrifuged to obtain the supernatant, 10 times its weight of 70% ethanol was added, and extracted under reflux at 70°C for 3 hours.

(5) The above extraction was repeated three times, the obtained extract was filtered, the obtained extract was filtered through a 0.45 μm membrane filter, and ethanol was removed using a reduced pressure concentrator to prepare a fermented arnica extract (sample AB-4).

<Comparative Example 1> Preparation of non-fermented arnica extract 1

(1) Arnica Montana and Bhuiamala were thoroughly washed, dried, and ground to a 40 mesh. The pulverized raw materials were mixed at a constant weight (100 g: 100 g).

(4') 70% ethanol (10 times the weight by weight) was added to the dried raw material and extracted under reflux at 70°C for 3 hours.

(5) The above extraction was repeated three times, the obtained extract was filtered, the obtained extract was filtered through a 0.45 μm membrane filter, and ethanol was removed using a reduced pressure concentrator to prepare a fermented arnica extract (sample AB-1).

<Comparative Example 2> Preparation of non-fermented arnica extract 2

(1) Arnica Montana and Bhuiamala were thoroughly washed, dried, and ground to a 40 mesh. The pulverized raw materials were mixed at a constant weight (100 g: 100 g).

(2) 70% ethanol, 10 times the weight by weight, was added to the dried raw material, and extraction was performed using a microwave extractor (Soxwave 100 product, stabilized after setting to 210W) at a frequency of 2,450 MHz for 15 minutes.

(5') The obtained extract was filtered through a 0.45 μm membrane filter, and ethanol was removed using a reduced pressure concentrator to prepare a fermented arnica extract (sample AB-2).

<Comparative Example 3> Preparation of non-fermented arnica extract 3

(1) Arnica Montana and Bhuiamala were thoroughly washed, dried, and ground to a 40 mesh. The pulverized raw materials were mixed at a constant weight (100 g: 100 g).

(2) 70% ethanol, 10 times the weight by weight, was added to the dried raw material, and extraction was performed using a microwave extractor (Soxwave 100 product, stabilized after setting to 210W) at a frequency of 2,450 MHz for 15 minutes.

(4") 10 times the weight of 70% ethanol was added to the ultrasonic extract and extracted under reflux at 70°C for 3 hours.

(5) The above extraction was repeated three times and the obtained extract was filtered. The obtained extract was filtered through a 0.45 μm membrane filter and ethanol was removed using a reduced pressure concentrator to prepare a fermented arnica extract (sample AB-3).

<Experimental example>

<Experimental Example 1> Cell viability evaluation test

Cell viability evaluation experiments on the samples obtained in Example 1 and Comparative Examples 1 to 3 were performed on macrophages, which play a central role in the immune response by delivering external antigen information to T cells and carrying out a cell-mediated immune response. This was performed using cells.

In this experiment, in order to confirm the cytotoxicity of Example 1 and Comparative Examples 1 to 3, the cell viability of Raw 246.7 cells, a large phagocytic cell, was confirmed at concentrations of 100 μg/ml, 500 μg/ml, and 1,000 μg/ml, respectively.

Specifically, RAW 264.7 cells, a mouse macrophage cell line, were cultured in DMEM (Dulbecco's modified Eagle's medium) containing 100 IU/mL penicillin, 100 IU/mL streptomycin, and 10% fetal bovine serum at 37°C in a 5% carbon dioxide incubator.

After dispensing RAW 264.7 cells into a 96-well plate at a concentration of 3x10 ⁴ cells/well, the cells were completely attached while culturing in a 5% carbon dioxide incubator at 37°C for 12 hours, and test substances (AB-1 to 4) were added. Diluted in PBS (phosphate buffered saline) and treated at concentrations of 0, 100, 500, and 1,000 μg/mL, 20 μL of MTT solution ((4,5-dimethylthiazol-2-yl)-2,5-diphenyl- tetrazolium bromide) and react for 4 hours. Afterwards, treat with 100 μL of DMSO, and after 1 hour, measure the absorbance at 540 nm using a microplate reader. Cell viability is compared using absorbance values. The evaluation results are shown in Figure 1 below.

As shown in Figure 1, the cytotoxicity of the test substances in macrophages was compared based on the cell viability (100%) of the untreated group, and the cell viability of all test substances was found to be at a concentration of 0 to 1,000 μg/ml. This was confirmed to be over 95%.

Therefore, it can be seen that not only Example 1 but also Comparative Examples 1 to 3 had no effect on cytotoxicity in macrophages.

<Experimental Example 2> NO concentration evaluation test

The samples obtained in Example 1 and Comparative Examples 1 to 3 were tested to determine whether the concentration ranged from 0 to 1,000 μg/ml had an effect on nitric oxide production ability (NO concentration). For reference, it is important to maintain the concentration of NO in the normal range because it causes inflammatory diseases when the concentration of NO is above the normal range. Therefore, NO production was induced in RAW 246.7 cells through LPS treatment by comparing the control group and the experimental group. The decrease in NO was confirmed.

Specifically, RAW 264.7 cells, a mouse macrophage cell line, were cultured in DMEM (Dulbecco's modified Eagle's medium) containing 100 IU/mL penicillin, 100 IU/mL streptomycin, and 10% fetal bovine serum at 37°C in a 5% carbon dioxide incubator.

After distributing RAW 264.7 cells to a 96-well plate at a concentration of 3x10 ⁴ cells/well, the cells were completely attached by culturing them in a 5% carbon dioxide incubator at 37°C for 12 hours at 1 μg, and 1 μg/μg of LPS, an inflammation-inducing substance, was added. mL and test substances (AB-1 to 4) were diluted in PBS (phosphate buffered saline), treated at concentrations of 0, 100, 250, and 500 μg/mL, and re-cultured for 24 hours. Obtain the upper liquid of the culture, add the same amount of Greek reagent (0.5% sulfanilamide, 2.5% phosphoric acid, and 0.5% naphthylethyleneamine), react for 10 minutes at room temperature, and measure the absorbance at 540 nm. The concentration of NO was calculated by comparing it with the standard curve of nitrite. The results are shown in Figure 2 below.

As shown in Figure 2 below, as a result of comparing the NO concentration of the control group and the experimental group (AB-1 to 4) treated with LPS in raw cell 246.7 cells for 24 hours, the control group was found to have the highest NO concentration, and the experimental group was found to have the highest NO concentration. It was confirmed that as the concentration of the test substance increased, the NO concentration tended to decrease.

The NO concentration decreased most significantly at 500 μg/ml in both Example 1 and Comparative Examples 1 to 3, and in particular, Example 1 was found to have the lowest concentration of 3.8 ± 0.7 μM, and among the comparative examples, Comparative Example 3, It was analyzed in the order of Comparative Example 2 and Comparative Example 1 as 5.0 ± 1.0 μM, 5.6 ± 0.5 μM, and 6.0 ± 0.4 μM, respectively.

<Experimental Example 3> Cytokine concentration evaluation test

Cytokines are signaling substances secreted when macrophages are activated by external antigens. The control group, which induced the production of inflammatory cytokines (IL-1β, IL-6, TNF-α) in RAW 246.7 cells through LPS treatment, was compared with the experimental group to determine whether the produced cytokines were reduced.

Specifically, the production of inflammation-related cytokines (IL-1β, IL-6, and TNF-α) in the cell culture medium was measured using an ELISA kit. Raw 264.7 cells were distributed in a 96-well plate at a concentration of 3x10 ⁴ cells/well, and cultured for 12 hours in a 5% carbon dioxide incubator at 37°C at 1 μg to ensure complete attachment of the cells and 1 μg/mL of LPS, an inflammation-inducing substance. and test substances (AB-1 to 4) were diluted in PBS (phosphate buffered saline) and treated at concentrations of 0, 100, 250, and 500 μg/mL and re-cultured for 24 hours. After collecting the supernatant, it is dispensed into a 96-well plate to which antibodies reactive to each cytokine are attached and reacted for 2 hours. After reaction, detection antibody, Sav-HRP reagent, and substrate solution were added, reaction stop solution was added, and measurement was performed at 450 nm using a microplate reader. The results are shown in Figure 3 below.

As shown in Figure 3 below, as a result of comparing the production amounts of L-1β, IL-6, and TNF-α after culturing the control group treated with LPS in Raw cell 246.7 cells and Example 1 and Comparative Examples 1 to 3 for 24 hours, The L-1β, IL-6, and TNF-α concentrations in the control group treated only with LPS were found to be the highest.

In both Example 1 and Comparative Examples 1 to 3, it was confirmed that as the amount of the test substance increased, the concentrations of L-1β, IL-6, and TNF-α tended to decrease.

The concentration of NF-α decreased most significantly at a concentration of 500 μg/ml, and in particular, in Example 1, it was confirmed to be the lowest concentration of 528.9 ± 39.9 μg/ml. Among the comparative examples, they were analyzed in the order of Comparative Example 3, Comparative Example 2, and Comparative Example 1 as 427.0±30.7 μg/ml, 549.2±15.4 μg/ml, and 528.9±19.3 μg/ml, respectively.

IL-1β and IL-6 production decreased most significantly at a concentration of 500 μg/ml, and in particular, in Example 1, the lowest concentrations were found to be 5.2 ± 1.2 μg/ml and 112.2 ± 15.0 μg/ml. Among the comparative examples, in the order of Comparative Example 3, Comparative Example 1, and Comparative Example 2, 6.5±1.3 μg/ml, 135.8±13.7 μg/ml, 8.9±1.1 μg/ml, 166.1±21.1 μg/ml, and 8.5±1 μg/ml, respectively. IL-1β was analyzed at 1.5 μg/ml and 155.9±22.3 μg/ml.

<Experimental Example 4> Arthritis severity assessment test in collagen-induced arthritis mice

The experimental material had a higher concentration of NO and decreased cytokines compared to the control group through cell experiments. Samples of Example 1 and Comparative Example 3 were selected and the severity of arthritis was evaluated using collagen-induced arthritis mice, a representative animal model method for rheumatoid arthritis.

The severity of arthritis was evaluated by comparing the occurrence of edema and redness in the toes, insteps, and ankles of mice from 3 to 6 weeks after arthritis was induced, and evaluating the incidence of arthritis, ankle thickness, and severity of symptoms as scores.

<Production of collagen-induced arthritis animal model>

Purchase 6-week-old Sprague-Dawley male white rats and manage them by acclimating them to the laboratory environment (temperature 22 ± 2°C, humidity 55 ± 5%, 12-hour light/dark cycle) by providing sufficient water and solid feed. Mix 150 μg of type 2 collagen per mouse with complete Freund's adjuvant at a concentration of 2 mg/mL in a 1:1 ratio and administer intradermally 1 cm below the base of the mouse's tail. Three weeks later, 100 μg of type 2 collagen and incomplete Freund's adjuvant were mixed 1:1 and administered intradermally 1 cm below the base of the mouse's tail to induce collagen induced arthritis (CIA).

The samples of Example 1 and Comparative Example 3 were orally administered at 500 mg/kg/day for a total of 6 weeks, and the control group was administered physiological saline solution. They were divided into 3 groups with 10 animals per group.

As a specific example, after the second injection of collagen-induced arthritis, the occurrence of redness and swelling in the two front and two hind paws of the experimental mice was observed with the naked eye once a week to evaluate the incidence of arthritis and the severity of arthritis in each period.

Severity assessment was assessed by an observer who was blinded to information about the experimental and control groups. The severity of arthritis for each leg was classified from 0 to 3 points. A score of 0 is a normal condition with no symptoms in the feet, a score of 1 is a condition where there is minimal swelling or redness in at least one place on the toes, top of the foot, or ankle, and a score of 2 is a condition where there is definite swelling in two or more of the toes, top of the foot, or ankle. A score of 3, where flushing occurred, was defined as a state in which significant edema and swelling were observed from the toes to the entire ankle area, or a state in which the foot could not be used freely due to stiffness. In mice, arthritis was considered to have occurred when the arthritis score was 2 or more. Ankle thickness was measured at the area with the most severe edema using an electrical caliper. The evaluation was conducted for 6 weeks. The results are shown in Figure 4 below.

As shown in Figure 4 below, both Example 1 and Comparative Example 3 showed a reduction in arthritis severity compared to the control group, and in particular, the severity of arthritis was reduced in the sample of Example 1 compared to the case of the sample of Comparative Example 3.

<Experimental Example 5> Cytokine concentration evaluation test in collagen-induced arthritis mice

ELISA was performed on inflammatory cytokines (IL-1β, IL-6, TNF-α) of the control group and test substances (AB-3, 4) using blood obtained by sacrificing collagen-induced arthritis mice that had completed the arthritis severity evaluation in Experimental Example 4. It was confirmed through .

Specifically, whole blood is obtained from an experimental animal, and then centrifuged at 4,000 rpm at 4°C for 10 minutes to separate serum. To evaluate the production of inflammation-related cytokines (IL-1β, IL-6, TNF-α) present in serum, they were measured using an ELISA kit. Dispense into a 96-well plate attached with antibodies reactive to each cytokine and react for 2 hours. After reaction, detection antibody, Sav-HRP reagent, and substrate solution were added, reaction stop solution was added, and measurement was performed at 450 nm using a microplate reader. The results are shown in Figure 5.

As shown in Figure 5 below, the concentrations of IL-1β, IL-6, and TNF-α in collagen-induced arthritis mice were analyzed to be lower than those in the control group in both Example 1 and Comparative Example 3. This trend was consistent with the cytokine concentration results of the cell experiment, and it was confirmed that cytokines were reduced through a decrease in the severity of arthritis in mice with collagen-induced arthritis.

Furthermore, the inflammatory cytokine (IL-1β, IL-6, TNF-α) content of Example 1 was 207.9 ± 22.6 pg/mL, 139.2 ± 25.5 pg/mL, and 21.9 ± 9.6 pg/mL, respectively, compared to the control group. It was confirmed that the 1β, IL-6, and TNF-α content decreased compared to 460.4±71.2 pg/mL, 586.4±82.2 pg/mL, and 107.7±26.7 pg/mL, and was further reduced compared to the sample of Comparative Example 1. was analyzed, and these results were consistent with the arthritis severity evaluation in Experimental Example 4 described above.

In conclusion, the ultrasonic extract using Arnica Montana and Bhuiamala contains Enterococcus faecium, Lactobacillus plantarum , Bifidobacterium longum , and lactobacilli. Bacillus acidophilus ( Lactobacillus acidophilus ), and When the powder or extract of a fermented product obtained by inoculating one or more types of fermentation strains selected from Leuconostoc mesenteroides is included as an active ingredient, the obtained composition may be used to treat sprains, bruises, muscle pain, joint pain, fracture pain, It was confirmed that it is suitable for applications that require improved anti-inflammatory and analgesic effects by simultaneously demonstrating the ability to treat back pain, stiff shoulders, neuralgia, rheumatic pain, skin itchiness, insect bites, and chilblains.

Claims (6)

An anti-inflammatory analgesic composition comprising as an active ingredient a powder of a fermented product obtained by inoculating a fermented strain of Enterococcus faecium into an ultrasonic extract using Arnica Montana and Bhuiamala , or an extract thereof. According to paragraph 1,
The powder or extract is an anti-inflammatory analgesic composition obtained by alcohol extraction of a fermented product. According to paragraph 2,
The alcohol extraction is an anti-inflammatory analgesic composition that is extracted at 60 to 70° C. for 2 to 3 hours under reflux. According to paragraph 1,
The ultrasonic extract is an anti-inflammatory analgesic composition extracted for 10 to 20 minutes at a frequency of 2200 to 2500 MHz. delete According to paragraph 1,
The anti-inflammatory analgesic is an anti-inflammatory analgesic composition that has the ability to treat sprains, bruises, muscle pain, joint pain, fracture pain, back pain, stiff shoulders, neuralgia, rheumatic pain, skin itchiness, insect bites, or chilblains.