KR102544441B1 - A novel anti-inflammatory evaluation method that treats Enterobacter aerogenes J2K-739 strain derived from skin flora and Enterobacter genus derived from skin flora as an irritant - Google Patents

Abstract

The present invention relates to an Enterobacter aerogenes J2K-739 strain derived from skin flora and a novel anti-inflammation evaluation method that treats Enterobacter genus derived from skin flora as an irritant and, more specifically, to an Enterobacter aerogenes J2K-739 strain derived from skin flora and a novel anti-inflammation evaluation method that treats Enterobacter genus derived from skin flora as an irritant, which uses an Enterobacter aerogenes J2K-739 strain derived from skin flora or a culture medium to induce inflammation to evaluate a material, instead of an anti-inflammation evaluation method using LPS to induce inflammation.

Description

A novel anti-inflammatory evaluation method that treats Enterobacter aerogenes J2K-739 strain derived from skin flora and Enterobacter genus derived from skin flora as an irritant {A novel anti-inflammatory evaluation method that treats Enterobacter aerogenes J2K-739 strain derived from skin flora and Enterobacter genus derived from skin flora as an irritant}

The present invention relates to a novel anti-inflammatory evaluation method for treating Enterobacter aerogenes J2K-739 strain derived from a skin flora and Enterobacter genus derived from a skin flora as an irritant, and more specifically, to an anti-inflammatory evaluation method Enterobacter aerogenes derived from a skin flora that evaluates the material by inducing inflammation using a J2K-739 strain or culture of Enterobacter aerogenes derived from a skin flora rather than a method for inducing inflammation due to LPS It relates to a novel anti-inflammatory evaluation method for treating strain J2K-739 and the genus Enterobacter derived from skin flora as irritants.

Human skin consists of the 'epidermis' that surrounds the outer surface of the skin, the 'dermis' that supplies the matrix that supports structures such as blood vessels and nerves under the epidermis, and the 'subcutaneous layer' that contains a large amount of fat between the dermis and muscles. organization is made up of.

Among them, it is known that the dermis contains about 70% of water, and the epidermis contains about 30% of water.

However, this is only a standard for the skin of a healthy person, and when the moisture content of the epidermis falls below 10%, negative effects such as roughening the skin and accelerating skin aging occur.

In addition, as dry skin is maintained, various inflammations such as skin troubles, psoriasis, and atopy may be induced.

Finally, as the frequency of exposure to external environmental factors such as fine dust, dry environment, and ultraviolet rays increases, demand for products with excellent skin barrier and moisturizing effects is increasing.

On the other hand, the inflammatory response induced through wounds or bacterial infection is a way to maintain homeostasis in vivo to restore damaged areas, and the inflammatory response accompanied by continuous stimulation induces cell death and lesions, which is deeply related to the onset of various diseases. there is.

Since macrophages are activated by cytokines and play a major role in the inflammatory response, drug efficacy methods using cell culture techniques are widely used.

Looking at the anti-inflammatory efficacy evaluation method at the cellular level, lipopolysaccharide (LPS) is administered to cultured macrophages to promote inflammatory mediating cytokine secretion and mass production of cyclooxygenase (COX) and nitric oxide (NO). treatment to evaluate the efficacy involved in inhibition.

Nevertheless, it is extremely rare for anti-inflammatory agents to prove their efficacy when applied to animal experiments.

These results suggest that, in terms of experimental techniques, it is important to conduct long-term experiments at the cell level to select effective candidates, and it is desirable to evaluate anti-inflammatory efficacy using small animals as the next step.

However, it has the disadvantages of a large number of small animals and high cost, so it is important to develop a new drug screening technique that provides an alternative.

Therefore, a rapid efficacy evaluation method is required at the cellular level, and an analysis using fluorescent labeling technology, which is an analysis technique capable of evaluating the efficacy of a drug in a non-invasive manner in the same subject, is required in animal experiments.

Rapid efficacy evaluation is possible when analyzing NO production from fluorescently labeled macrophages, and if cell mobility is analyzed through a bio-image fluorescence analyzer, it is easy to observe macrophage mobility in real time, allowing in vivo testing without sacrificing animals or fixing living specimens. By tracking the efficacy of the drug, a practical means for screening the drug efficacy can be presented.

In particular, the technique of interpreting this in real time is a technique capable of providing information on the temporal pharmacokinetics of the treatment drug by analyzing the onset time and extinction time of the drug's efficacy.

However, since these methods frequently show self-luminescence in vivo, it is necessary to establish accurate experimental techniques.

Accordingly, a clinical evaluation method for a cosmetic composition for improving skin inflammation, in which the subject's burden for the side effects of the test is small, the application of the cosmetic is consistently performed by the evaluator, and the evaluation period is short and objective results can be derived. there is a demand for

Korean Patent Registration No. 10-2134930

The present invention has been researched and developed to solve the above problems, and an object of the present invention is to enterobacter aerogenes J2K-739 strain derived from a skin flora, rather than an anti-inflammatory evaluation method that induces inflammation due to LPS. Alternatively, a novel anti-inflammatory evaluation method of treating Enterobacter aerogenes J2K-739 strain derived from a skin flora and Enterobacter genus derived from a skin flora as a stimulant to evaluate the material by inducing inflammation using the culture want to provide

The novel anti-inflammatory evaluation method of the present invention, which treats Enterobacter genus derived from skin flora as an irritant,

A cell culture step of culturing RAW 264.7 cells (S100); and

Subculture step (S200) of dispensing the RAW 264.7 cells and stabilizing them through a wet incubator; and

A skin flora reaction step (S300) of treating a sample in a medium, treating a strain or culture derived from the skin flora, and reacting for a certain period of time; and

After the reaction, a reagent mixing step (S400) for mixing the same amount of Griess reagent with the cell culture supernatant; and

After providing the mixed mixture to the well plate, a reagent reaction step (S500) for reacting for 10 minutes; and

Absorbance measurement step (S600) for obtaining the reacted mixture and measuring absorbance at 540 nm; by including, the object of the present invention is solved.

As can be seen through the present invention, the anti-inflammatory evaluation method is not a method of inducing inflammation due to LPS, but by using Enterobacter aerogenes J2K-739 strain or culture derived from skin flora to induce inflammation By evaluating the material, it is possible to evaluate simply, quickly and efficiently under in vitro conditions.

The evaluation method of the present invention can be quantitatively evaluated simply and quickly, so it is suitable for quality control at each production stage and final product quality control for cosmetic development.

Therefore, the evaluation method of the present invention, based on the measurement results of NO (nitric oxide) production changes related to inflammatory response, immune response, skin aging, skin regeneration, and various diseases, can be applied to the field of diagnosis and treatment. will be.

1 is a 16S rRNA sequence diagram of the identification result of Enterobacter aerogenes J2K-739 strain derived from the skin flora of the present invention.
Fig. 2 is a schematic diagram of experimental progress showing a novel anti-inflammatory evaluation method in which Enterobacter genus derived from skin flora is treated as an irritant.
3 is experimental data results of a novel anti-inflammatory evaluation method in which enterobacter genus derived from skin flora is treated as an irritant.
4 is a certificate of deposit demonstrating the deposit of the Enterobacter aerogenes J2K-739 strain derived from the skin flora of the present invention.

Hereinafter, the present invention will be described.

The term 'composition' in the present invention preferably means a composition for external application for skin, and the composition for external application for skin generally means all compositions applied to the skin, and includes pharmaceuticals, quasi-drugs, cosmetics, and the like.

On the other hand, in the present invention, the meaning of "included as an active ingredient" means that the culture filtrate is added to the composition to the extent that it can exhibit the effect of improving skin conditions from the composition of the present invention, and for ingredient delivery and stabilization, etc. It is meant to include formulating in various forms by adding various ingredients as subcomponents.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are merely exemplary descriptions of the present invention, and the scope of the present invention is not limited to these examples.

In order to solve the above problems, it was attempted to find common flora harmful to the skin by isolating and identifying skin flora from the skin.

Specifically, a facial skin sample of a healthy woman was taken from the Semyung University Clinical Center, and the strain was isolated and used as a sample.

The skin samples were suspended and diluted in sterile physiological saline, respectively, and 0.1 mL each was spread on Difco Tryptic Soy Agar medium.

Then, the plate was incubated for 2 days in a 35° C. incubator.

Thereafter, each of the resulting colonies was streak-inoculated on a TSA plate, and cultured at 35° C. for 2 days to obtain colonies showing off-white colonies.

In the same manner as above, various types of skin flora were isolated and identified.

For example, the above skin flora,

Bacteria of the genus Enterobacter, bacteria of the genus Klebsiella, Staphylococcus aureus, Bacillus licheniformis, Moraxella osloensis, and Bacillus subtilis characterized by

At this time, in the example of the present invention, bacteria of the genus Enterobacter were applied, and it is obvious that any of the various bacteria described above can be applied.

In the present invention, it was confirmed through an in vitro experiment that inflammation can be induced by treating the fermented filtrate of the normal flora, and through this, a novel anti-inflammatory evaluation method was developed.

This will be explained in detail below.

In the present invention, Enterobacter aerogenes J2K-739 strain with accession number KCTC 14920BP derived from the skin flora was obtained.

At this time, it is characterized in that the material is evaluated by inducing inflammation using the Enterobacter aerogenes J2K-739 strain or culture thereof having accession number KCTC 14920BP derived from the skin flora.

At this time, preferably, the material corresponds to a cosmetic composition.

In addition, as shown in FIG. 2, the novel anti-inflammatory evaluation method for treating Enterobacter genus derived from the skin flora as an irritant,

A cell culture step of culturing RAW 264.7 cells (S100); and

Subculture step (S200) of dispensing the RAW 264.7 cells and stabilizing them through a wet incubator; and

A skin flora reaction step (S300) of treating a sample in a medium, treating a strain or culture derived from the skin flora, and reacting for a certain period of time; and

After the reaction, a reagent mixing step (S400) for mixing the same amount of Griess reagent with the cell culture supernatant; and

After providing the mixed mixture to the well plate, a reagent reaction step (S500) for reacting for 10 minutes; and

It is characterized in that it comprises a; absorbance measuring step (S600) for obtaining the reacted mixture and measuring absorbance at 540 nm.

The above process will be described in detail through examples.

In general, as an experimental method for inducing inflammation due to LPS, a NO assay experimental method was used.

Specifically, the amount of NO produced from RAW 264.7 cells was measured using Griess' reagent for NO2- present in the cell culture medium.

RAW 264.7 cells were adjusted to 1×10 ⁴ cells/well using DMEM (10% FBS) medium, then inoculated into a 96-well plate and cultured in a humidified incubator at 37° C. and 5% CO ₂ .

After serial dilution of the sample, the cells were treated with 1 μg/mL of LPS and cultured for 24 h.

Cell culture supernatant and the same amount of Griess reagent were mixed and reacted for 10 min in 96 well plates, and absorbance was measured at 540 nm.

The method of inducing inflammation described above is quite complicated, and considerable difficulties have occurred in selecting clinical subjects.

Example 1: Preparation of Enterobacter aerogenes J2K-739 culture

Seed culture of the pure isolated Enterobacter aerogenes J2K-739 strain was used by inoculating a single colony in TSB medium and culturing it in a 35° C. incubator for 24 hours.

The composition of the medium optimized for the growth of the Enterobacter aerogenes J2K-739 strain is an optimized medium containing 2.5% glucose, 2.0% yeast extract, 0.5% sodium chloride, and 0.25% potassium dibasic phosphate, pH After inoculating the seed culture medium to 5% (V / V) in the medium adjusted to 7.5, it was cultured for 48 hours at a temperature of 35 ° C and a rotation speed of 150 rpm.

After culturing, cells were removed from the culture medium by centrifugation, and the supernatant was filtered to obtain an Enterobacter aerogenes J2K-739 culture from which the cells were completely removed.

Comparative Example 1: Preparation of Enterobacter aerogenes J2K-727 culture

It was prepared in the same way as in Example 1.

Comparative Example 2: Preparation of Enterobacter aerogenes J2K-753 culture

It was prepared in the same way as in Example 1.

Comparative Example 3: Preparation of Enterobacter aerogenes J2K-743 culture

It was prepared in the same way as in Example 1.

Comparative Example 4: Preparation of Klebsiella pneumoniae J2K-755 culture

It was prepared in the same way as in Example 1.

strain number strain name age gender J2K-739 Enterobacter aerogenes 51 female J2K-727 Enterobacter aerogenes 52 female J2K-753 Enterobacter aerogenes 48 female J2K-743 Enterobacter aerogenes 26 female J2K-755 Klebsiella pneumoniae 52 female

Examples and Comparative Examples were prepared in the same way, and as shown in Table 1, the difference was that facial skin samples of healthy women were taken and strains were separated and used as each sample.

Experimental Example 1: NO assay test method

Anti-inflammatory evaluation method of the present invention,

A cell culture step of culturing RAW 264.7 cells (S100); and

Subculture step (S200) of dispensing the RAW 264.7 cells and stabilizing them through a wet incubator; and

A skin flora reaction step (S300) of treating a sample in a medium, treating a strain or culture derived from the skin flora, and reacting for a certain period of time; and

After the reaction, a reagent mixing step (S400) for mixing the same amount of Griess reagent with the cell culture supernatant; and

After providing the mixed mixture to the well plate, a reagent reaction step (S500) for reacting for 10 minutes; and

It is characterized in that it comprises a; absorbance measuring step (S600) for obtaining the reacted mixture and measuring absorbance at 540 nm.

Based on this, an experimental example was established as follows.

For example, the amount of NO produced from RAW 264.7 cells was measured using Griess reagent NO ₂ - present in the cell culture medium.

RAW 264.7 cells were cultured using DMEM (10% FBS) medium, and the culture conditions were 37℃, 5% CO ₂ , 24 hours. (S100)

Then, after adjusting to 1×10 ⁴ cells/well, it was inoculated into a 96-well plate and cultured in a humidified incubator at 37°C and 5% CO ₂ (S200).

Thereafter, the samples were serially diluted and treated with cells, and then treated with Example 1 and Comparative Examples 1 to 4 and cultured for 24 hours. (S300)

Thereafter, the cell culture supernatant and the same amount of Griess reagent were mixed (S400), reacted in 96 well plates for 10 minutes (S500), and the absorbance was measured at 540 nm (S600).

Thereafter, the stimulant treatment group (Example 1) and comparative examples were compared and analyzed.

The above experimental results are shown in FIG. 3, and as shown in FIG. 3, the horizontal axis is a value according to each embodiment and concentration, and the vertical axis is related to the amount of expression.

It was found that Example 1 increased the production of nitric oxide in a concentration-dependent manner at a concentration of 1 to 10%.

Compared to Comparative Examples 1 to 4, it was found that the production of nitric oxide was significantly increased, and at the concentration of 10% in Example 1, the production of nitric oxide increased by about 1.2 times compared to the inflammation caused by the conventional LPS.

Therefore, it is judged that the 10% concentration of Example 1 is the most preferable evaluation.

From the above description, those skilled in the art to which this application pertains will be able to understand that this application can be implemented in other specific forms without changing its technical spirit or essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present application should be construed as including all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts rather than the detailed description above.

Korea Research Institute of Bioscience and Biotechnology KCTC14920BP 20220322

<110> J2KBIO CO., LTD. <120> A novel anti-inflammatory evaluation method that treats Enterobacter aerogenes J2K-739 strain derived from skin flora and Enterobacter genus derived from skin flora as an irritant <130> J2K-739 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 778 <212> RNA <213> Enterobacter aerogenes <400> 1 gagcggcgga cgggtgagta atgtctggga aactgcctga tggaggggga taactactgg 60 aaacggtagc taataccgca taacgtcgca agaccaaagt gggggacctt cgggcctcat 120 gccatcagat gtgcccagat gggattagct agtaggtggg gtaatggctc acctaggcga 180 cgatccctag ctggtctgag aggatgacca gccacactgg aactgagaca cggtccagac 240 tcctacggga ggcagcagtg gggaatattg cacaatgggc gcaagcctga tgcagccatg 300 ccgcgtgtat gaagaaggcc ttcgggttgt aaagtacttt cagcgaggag gaaggcatta 360 aggttaataa ccttggtgat tgacgttact cgcagaagaa gcaccggcta actccgtgcc 420 agcagccgcg gtaatacgga gggtgcaagc gttaatcgga attactgggc gtaaagcgca 480 cgcaggcggt ctgtcaagtc ggatgtgaaa tccccgggct caacctggga actgcattcg 540 aaactggcag gctagagtct tgtagagggg ggtagaattc caggtgtagc ggtgaaatgc 600 gtagagatct ggaggaatac cggtggcgaa ggcggcccccc tggacaaaga ctgacgctca 660 ggtgcgaaag cgtggggagc aaacaggatt agataccctg gtagtccacg ccgtaaacga 720 tgtcgacttg gaggttgtgc ccttgaggcg tggcttccgg agctaacgcg ttaagtcg 778 <210> 2 <211> 673 <212> RNA <213> Enterobacter aerogenes <400> 2 ggttgtgccc ttgaggcgtg gcttccggag ctaacgcgtt aagtcgaccg cctggggagt 60 acggccgcaa ggttaaaact caaatgaatt gacgggggcc cgcacaagcg gtggagcatg 120 tggtttaatt cgatgcaacg cgaagaacct tacctactct tgacatccag agaacttagc 180 agagatgctt tggtgccttc gggaactctg agacaggtgc tgcatggctg tcgtcagctc 240 gtgttgtgaa atgttgggtt aagtcccgca acgagcgcaa cccttatcct ttgttgccag 300 cgatccggtc gggaactcaa aggagactgc cagtgataaa ctggaggaag gtggggatga 360 cgtcaagtca tcatggccct tacgagtagg gctacacacg tgctacaatg gcatatacaa 420 agagaagcga cctcgcgaga gcaagcggac ctcataaagt atgtcgtagt ccggattgga 480 gtctgcaact cgactccatg aagtcggaat cgctagtaat cgtagatcag aatgctacgg 540 tgaatacgtt cccgggcctt gtacacaccg cccgtcacac catgggagtg ggttgcaaaa 600 gaagtaggta gcttaacctt cgggagggcg cttaccactt tgtgattcat gactggggtg 660 aagtcgtaac agg 673

Claims (7)

Using the Enterobacter aerogenes J2K-739 strain or culture thereof of accession number KCTC 14920BP derived from skin flora,
A novel anti-inflammatory evaluation method for treating the skin flora-derived Enterobacter genus as an irritant, characterized in that the material is evaluated by inducing inflammation.

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