KR102059411B1 - A Atopy Treating Composition Using A Stem Cell Crushed Extract(Non-shell Stem Cell) and Wormwood Extract and a A Cosmetic Using thereof - Google Patents

Abstract

The present invention relates to a composition for treating atopic dermatitis using a stem cell lysate extract (non-shell stem cells) and a wormwood extract, and to cosmetics using the same. The composition for treating atopic dermatitis comprises a stem cell lysate extract and a wormwood extract.

Description

A Atopy Treating Composition Using A Stem Cell Crushed Extract (Non-shell Stem Cell) and Wormwood Extract and a A Cosmetic Using Herb}

The present invention relates to a composition for atopic dermatitis and cosmetics using the same, and more particularly, it is possible to increase the yield by culturing a large amount of stem cells, to increase the yield of the active ingredient in stem cells, and to implement the characteristics of the mugwort extract on the skin It relates to a composition for atopic skin using stem cell crush extract and mugwort extract and cosmetics using the same.

Stem cells are undifferentiated cells with the ability to differentiate into various types of body tissues, and many studies have been conducted using them because of their ability to differentiate into various tissue cells. Among stem cells, adult stem cells can be easily obtained from areas such as fat, bone marrow, umbilical cord blood or placenta, and have fewer ethical problems than embryonic stem cells. It is done.

Treatment for injecting adult stem cells directly is limited to self-treatment for reasons such as immune rejection. To overcome the limitations of this self-treatment, one method would be to destroy the stem cells to destroy the immunogenic cell membranes that cause immune rejection. The inventors of the present invention have established the concept of the stem cell lysate extract for the first time and applied for the original patent. The advantage of the original patent is the universality that can be used by anyone by removing the cell membrane attached to the immunogenic reaction causing the immune rejection reaction, the reason for mass cultivation is established, and as a result, the popularization and industrialization of stem cells can be achieved.

Therefore, in order to achieve the popularization and industrialization of stem cells, continuous research and development of a method useful for mass culturing stem cells and a method of extracting the active ingredient thereof is required.

Patent Publication No. 10-2013-0118674

In order to solve the above problems, an object of the present invention is to cultivate a large amount of stem cells, to increase the yield of cells, skin sensitization reaction of the stem cell crush extract and skin sensitization of wormwood extract is reduced and skin It relates to a composition for atopic skin using stem cell crush extract and mugwort extract with increased defense and cosmetics using the same.

In order to achieve the above object, the composition for atopic skin using the stem cell crush extract and mugwort extract of the present invention is characterized in that it comprises a stem cell crush extract and wormwood extract.

80 parts by weight of the stem cell crush extract is characterized in that the 20 parts by weight of the wormwood extract is mixed.

And, another embodiment of the present invention is characterized in that the cosmetic is made of the composition of the above-described configuration.

Atopic dermatological composition using the stem cell crush extract and mugwort extract according to the present invention and cosmetics using the same has the following effects.

There is an advantage that the function of the anti-inflammatory and skin improvement through the performance factor of the stem cell crush extract, and the function of skin soothing and skin purification through the mugwort extract is maximized.

1 is a view showing a stem cell active ingredient three bottom extraction apparatus according to the present invention.
2 is to determine the anti-arthritis effect of the stem cell crush extract according to the present invention Drawing to confirm that the chondrocytes by using a specific marker of chondrocytes CD44.
FIG. 3 is a diagram illustrating the toxicity of a substance to cells by measuring stem cell crushing component extracts on chondrocytes for 24 hours of culture and measuring cell viability.
4 is treated with IL-1α on chondrocytes to induce inflammation, and treatment of stem cell lysate extracts by concentration on the chondrocytes causing inflammation confirms inhibition of NO production as an inflammation mediator.
5 is a view confirming the effects of inhibiting the production of NO induced after the treatment of stem cell lysate extracts induced chondrocytes induced by IL-1α and iNOS and COX-2 expression.
Figure 6 confirms that chondrocytes treated with stem cell lysate extract inhibit the migration of NF-κB protein by IL-1α into the nucleus.
Figure 7 by treating the stem cell crush extract to chondrocytes inflamed by IL-1α Figures confirming iMAPKs activity.
8 is a diagram confirming the anti-inflammatory effect of the present stem cell extract through genetic expression.
9 is a diagram confirming the cartilage forming effect through the gene of sox-9 involved in the development of cartilage formation.
10 is a view showing the expression of significant cytotoxicity as a result of treatment of the stem cell lysate extract according to the present invention by concentration.
11 is a diagram showing the expression of cytotoxicity as a result of treating the stem cell lysate extract according to the present invention simultaneously with LPS.
12 is a view showing a change in cell morphology of the stem cell lysate extract according to the present invention treated with LPS.
Figure 13 shows the change in the concentration of nitric oxide as a result of treating the stem cell lysate extract according to the present invention with LPS.
Figure 14 shows the iNOS and COX-2 protein expression changes as a result of treatment with stem cell lysate extract LPS according to the present invention.
Figure 15 shows the change in TNFα as a result of treatment of LPS with stem cell lysate extract according to the present invention
Figure 16 shows the changes in IL-1β as a result of treatment with stem cell lysate extract LPS according to the present invention.
Figure 17 is a graph showing the effect of the mixture containing the stem cell crush extract and mugwort extract according to the present invention on the skin moisturizing.
18 is a graph showing the effect of the mixture containing stem cell crush extract and mugwort extract according to the present invention on the skin regeneration factor (FGF-2).
19 is a graph showing the effect of the mixture containing the stem cell crush extract and mugwort extract according to the present invention on the skin regeneration factor (VEGF).
20 is a graph showing the effect of the mixture containing stem cell crush extract and mugwort extract according to the present invention on the skin regeneration factor (TIMP-1).
Figure 21 is a graph showing the effect of the mixture containing the stem cell crush extract and mugwort extract according to the present invention on the atopic dermatitis severity index (Skorad index).

Hereinafter, with reference to the accompanying drawings, a preferred embodiment of the composition for atopic skin using stem cell lysate extract and mugwort extract according to the present invention and cosmetics using the same will be described in detail.

First, the stem cell active ingredient extracting apparatus of the present invention, as shown in Figure 1, the body portion 10 and the inside is formed in the space and the interior is blocked, the space is provided inside the body portion 10 and upwards It is formed in a relatively small size with respect to the opening of the first container portion 12 and the first container portion 12, is provided in the first container portion 12, the upper side is opened to insert the stem cells therein The second vessel portion 14 to be broken, the crushed portion 16 to crush the stem cells in the second vessel portion 14, and the valve 18 to control the flow of air into the body portion 10 And, it may be configured to include a pump 20 for sucking the air in the body portion 10.

First, the present invention extraction device is provided with a body portion (10). As shown in Figure 1, the body portion 10 is provided with a sealed space therein to block the outside and the inside.

Inside the body portion 10, a first vessel portion 12 is provided. The first container portion 12 is formed so that a space is provided therein and an upper portion thereof is opened. The first container portion 12 is loaded with ice to serve to lower the temperature of the stem cells in the second container portion 14 to be described below.

The second container part 14 is provided inside the first container part 12. The second container portion 14 is formed such that the space is provided inside and the upper portion is opened similarly to the first container portion 12. In particular, the second container part 14 may be formed in a relatively smaller size than the first container part 12, and the second container part 14 may be loaded in the first container part 12. have. Stem cells may be loaded in the second container part 14.

And, the present invention may be further provided with a crushing portion 16. The crushing unit 16 serves to crush the stem cells loaded in the second container unit 14 using ultrasonic waves.

In addition, the present invention is provided with a flow path through which the outside and air flow, and a valve 18 for selectively blocking the flow of air through the flow path is provided. The other end of the flow path is provided with a pump 20 to discharge the air inside the body portion 10 to the outside to make the inside of the body portion 10 in a vacuum state.

In addition, the stem cells introduced into the second container may be put in a stock solution so that the stem cells are expanded. That is, the stem cells are put in a stock solution such as distilled water so that the stem cells are expanded by osmotic pressure so that they can be broken by a small impact.

The medium composition for stem cell mass culture and the medium composition method of the present invention will be described in detail.

In the present invention, a new medium is prepared to increase cell yield, and when the cells are crushed, more effective ingredients can be obtained.

The medium is a known medium commonly used in the culture of animal cells, for example, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basic Medium Eagle (BME), RPMI 1640, F-10, F12, DMEM-F12, α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), MacCoy's 5A Medium, AmnioMax, AminoMaxII complete Medium, Chang's Medium MesemCult-XF Medium, etc., but is not particularly limited thereto.

Then, an additive composition is added to the medium. The additive composition is hyaluronic acid, glycine, histidine, isoleucine, methionine, phenylalanine, proline, hydroxyproline, serine, threonine, tryptophan, tyrosine, valine, bFGF, EGF, VEGF, KGF, HGF, TGF, vitamin C, vitamin B1 And vitamin B12, vitamin E, selenium and transferrin.

First, hyaluronic acid is included in the additive composition. The hyaluronic acid serves as a scaffold during cell culture, and may be included at a concentration of 10 µg / ml with respect to the medium composition.

In addition, the additive composition includes glycine. The glycine is a amino acid that serves as a nutrient to the growth of the cell, it may be included in a concentration of 1ng / ㎖ with respect to the medium composition.

The additive composition includes histidine. The histidine is an amino acid, which serves as a nutrient for cell growth, and may be included at a concentration of 1 ng / ml relative to the medium composition.

The additive composition includes isoleucine. The isoleucine is an amino acid, which serves as a nutrient for cell growth, and may be included at a concentration of 1 ng / ml relative to the medium composition.

The additive composition includes methionine. The methionine is an amino acid, which serves as a nutrient for cell growth, and may be included at a concentration of 1 ng / ml relative to the medium composition.

The additive composition includes phenylalanine. The phenylalanine is an amino acid, which serves as a nutrient for cell growth, and may be included at a concentration of 1 ng / ml relative to the medium composition.

The additive composition includes proline The proline is an amino acid, which serves as a nutrient for cell growth, and may be included at a concentration of 10 ng / ml relative to the medium composition.

The additive composition includes hydroxyproline. The hydroxyproline is an amino acid, which serves as a nutrient for cell growth, and may be included at a concentration of 5 ng / ml relative to the medium composition.

The additive composition includes serine The serine is an amino acid, which serves as a nutrient for cell growth, and may be included at a concentration of 1 ng / ml relative to the medium composition.

The additive composition includes threonine. The threonine is an amino acid, which serves as a nutrient for cell growth, and may be included at a concentration of 1 ng / ml relative to the medium composition.

The additive composition includes tryptophan The tryptophan is an amino acid that serves as a nutrient for cell growth, and may be included at a concentration of 1 ng / ml relative to the medium composition.

The additive composition includes tyrosine. The tyrosine is an amino acid, which serves as a nutrient for cell growth, and may be included at a concentration of 1 ng / ml relative to the medium composition.

The additive composition includes valine The valine is an amino acid, which serves as a nutrient for cell growth, and may be included at a concentration of 2 ng / ml relative to the medium composition.

In addition, the additive composition includes bFGF. The bFGF helps to grow the cells to be cultured, and may be included at a concentration of 9 μg / ml relative to the medium composition.

The additive composition includes EGF. The EGF helps growth of the cultured cells, and may be included at a concentration of 1.5 μg / ml relative to the medium composition.

The additive composition includes VEGF. The VEGF helps growth of the cultured cells, and may be included at a concentration of 1 μg / ml relative to the medium composition.

The additive composition includes KGF. The KGF helps growth of the cultured cells, and may be included at a concentration of 1.2 μg / ml relative to the medium composition.

The additive composition includes HGF. The HGF helps to grow the cultured cells, and may be included at a concentration of 0.5 μg / ml relative to the medium composition.

The additive composition includes TGF. The TGF helps to grow the cultured cells, and may be included at a concentration of 0.5 μg / ml relative to the medium composition.

In addition, the additive composition includes vitamin C. The vitamin C assists the growth of the cultured cells, and may be included at a concentration of 2 μg / ml relative to the medium composition.

The additive composition includes vitamin B1. The vitamin B1 assists the growth of the cultured cells, and may be included at a concentration of 0.5 μg / ml relative to the medium composition.

The additive composition includes vitamin B12. The vitamin B12 helps to grow the cells to be cultured, and may be included at a concentration of 3 μg / ml relative to the medium composition.

The additive composition includes vitamin E. The vitamin E assists the growth of the cultured cells, and may be included at a concentration of 500 μg / ml relative to the medium composition.

In addition, the additive composition includes selenium. The selenium is to help the activity of the cell, it may be included in a concentration of 1.8 μg / ㎖ with respect to the medium composition.

The additive composition includes transferrin. The transferrin is to help the cell activity, it may be included in a concentration of 12㎛ / ㎖ with respect to the medium composition.

Next, a method for preparing a cell culture medium composition, a method for preparing a cell culture medium composition, and a method for preparing a stem cell lysate extract using the stem cell active ingredient 3 low extraction method using the stem cell active ingredient extracting apparatus according to the present invention will be described. .

Step 1: Stem cells are extracted from any one of human body fat, bone marrow, umbilical cord blood or placenta. In the present invention, the fat is collected by liposuction and then purified by enzyme treatment and centrifugation several times to separate fat stem cells.

Second step: The isolated stem cells are cultured using a medium made using the above-described mass culture medium composition of the present invention. When the stem cell mass culture medium composition is used, the growth rate of stem cells is increased to increase the yield of stem cells.

Third step; After removing the culture medium in which the stem cells have been cultured, the stem cells are separated, and cell suspension is obtained and passaged.

Step 4: Once the cells have grown to sufficient density, remove the cells from the plate. Stem cells are isolated and washed several times using saline or PBS (phosphate buffer saline). Cell counting is used to measure the number of cells and to produce a constant concentration of suspension.

The fifth step; Remove supernatant and add extender to allow stem cell membrane to break. Ultrasonic wave is added and the cells are crushed by the stem cell active ingredient 3 low extraction method. When the microscope confirms that all the stem cells are broken, the shredding is terminated. Through the process of the three low extraction method, the effective cell membrane disruption in a short time to maintain most of the active ingredients such as growth factors and cell active material inside the stem cells.

Step 6: The hypotonic extender and stem cell disruption mixtures contain cell membrane residues along with various growth factors and cell activators that are the stem cell contents. Unnecessary cell membrane residues are removed by centrifugation and microfilter. At this time, the immunogenicity causing the immunorejection reaction attached to the cell membrane is removed, and the stem cell active ingredient that anyone can use is extracted.

Seventh step; Stem cell crushed extract made through the above freeze storage or freeze-dried storage and use.

The active ingredients obtained through the above steps are called stem cell lysate extracts, also known as shelled stem cells.

Hereinafter, the results of experiments by extracting the effective extract of the crushed extract of stem cells using the stem cell mass culture medium composition by the additive composition according to the present invention.

Comparative Example 1

11,12 parts by weight of FBS was added to 100 parts by weight of Dulbecco's Modified Eagle Medium (DMEM), and cultured with stem cells in a medium containing 0.6 parts by weight of penicillin as an antibiotic. Factors were extracted.

Example 2

Stem cells are cultured in a medium mixed with 20 parts by weight of serum containing nutrients without 100 parts of Dulbecco's Modified Eagle Medium (DMEM), and grown according to the above-described procedure. Extracted.

Example 3

Stem cells were cultured in a medium mixed with 5 parts by weight of the additive composition of the present invention shown in Table 1 in 100 parts by weight of DMEM (Dulbecco's Modified Eagle Medium), and grown according to the above-described procedure, and extracted the effective factors.

Example 4

Stem cells were cultured in a medium in which 10 parts by weight of the additive composition of the present invention shown in Table 1 was mixed with 100 parts by weight of DMEM (Dulbecco's Modified Eagle Medium), and grown according to the above-described procedure, and the effective factors were extracted.

Example 5

Stem cells were cultured in a medium mixed with 15 parts by weight of the additive composition of the present invention shown in Table 1 to 100 parts by weight of DMEM (Dulbecco's Modified Eagle Medium), and grown according to the above-described procedure, and extracted the effective factors.

Example 6

Stem cells were cultured in a medium mixed with 20 parts by weight of the additive composition of the present invention shown in Table 1 in 100 parts by weight of DMEM (Dulbecco's Modified Eagle Medium), and grown according to the above-described procedure, and extracted the effective factors.

Example 7

Stem cells were cultured in a medium mixed with 25 parts by weight of the additive composition of the present invention shown in Table 1 to 100 parts by weight of DMEM (Dulbecco's Modified Eagle Medium), and grown according to the above-described procedure, and extracted the effective factors.

Example 8

Stem cells were cultured in a medium mixed with 30 parts by weight of the additive composition of the present invention shown in Table 1 to 100 parts by weight of DMEM (Dulbecco's Modified Eagle Medium), and grown according to the above-described procedure, and extracted the effective factors.

Example 9

Stem cells were cultured in a medium mixed with 35 parts by weight of the additive composition of the present invention shown in Table 1 in 100 parts by weight of DMEM (Dulbecco's Modified Eagle Medium), and grown according to the above-described procedure, and extracted the effective factors.

Example 10

Stem cells were cultured in a medium mixed with 40 parts by weight of the additive composition of the present invention shown in Table 1 in 100 parts by weight of DMEM (Dulbecco's Modified Eagle Medium), and grown according to the above-described procedure, and extracted the effective factors.

Example 11

Stem cells were cultured in a medium mixed with 45 parts by weight of the additive composition of the present invention shown in Table 1 to 100 parts by weight of DMEM (Dulbecco's Modified Eagle Medium), and grown according to the above-described procedure, and extracted the effective factors.

Example 12

Stem cells were cultured in a medium mixed with 50 parts by weight of the additive composition of the present invention shown in Table 1 to 100 parts by weight of DMEM (Dulbecco's Modified Eagle Medium), and grown according to the above-described procedure, and extracted the effective factors.

Classification Kinds density Scaffold Hyaluronic acid 10 Μg / ml amino acid Glycine One ng / ml Histidine One ng / ml Isoleucine One ng / ml Methionine One ng / ml Phenylalanine One ng / ml Proline 10 ng / ml Hydroxyproline 5 ng / ml Serine One ng / ml Threonine One ng / ml Tryptophan One ng / ml Tyrosine One ng / ml Valine 2 ng / ml Growth factor bFGF 9 Μg / ml EGF 1.5 Μg / ml VEGF One Μg / ml KGF 1.2 Μg / ml HGF 0.5 Μg / ml TGF 0.5 Μg / ml vitamin Vitamin C 2 Μg / ml Vitamin B1 0.5 Μg / ml Vitamin B12 3 Μg / ml Vitamin E 500 Μg / ml Trace elements Selenium 1.8 ng / ml Transferrin 12 Μg / ml

Next, the experimental results comparing the amounts of the cells obtained as a result of culturing the cells by the cell culture medium composition according to the present invention will be described.

Measurement form Obtained before cell disruption Detection amount (average) 0 group Cell yield (pieces / ml) Control 1.1x10 ^ 4 Comparative Example 1 2.3x10 ^ 5 Example 1 8.7x10 ^ 5 Example 2 8.6x10 ^ 5 Example 3 8.9x10 ^ 5 Example 4 9.3x10 ^ 5 Example 5 9.7x10 ^ 5 Example 6 1.2x10 ^ 6 Example 7 1.5x10 ^ 6 Example 8 1.8x10 ^ 6 Example 9 2.1x10 ^ 6 Example 10 2.4x10 ^ 6 Example 11 2.5x10 ^ 6 Example 12 2.5x10 ^ 6

As shown in Table 2, it can be confirmed that the cell yield is high as a result of Examples 2 to 12. However, in the case of Example 2 and approximate to Example 1 injecting the serum, Example 11 and Example 12 can be confirmed that the increase efficiency is relatively low, the yield efficiency is low.

Next, the results of crushing the cells obtained as a result of culturing the cells by the cell culture medium composition according to the present invention will be described experimental results comparing the detection amount of the active ingredient. In particular, compared to Example 1, Example 6 in which the additional amount of the additive composition was the same was compared.

Measurement form Detection amount (average) group Control Comparative Example 1 Example 1 Example 6 After cell disruption Total protein (mg / ml) 50.48 ± 1.07 181.12 ± 1.45 961.53 ± 2.03 2002.13 ± 2.82 After cell disruption TGF (pg / ml) 13.06 ± 1.85 113.76 ± 2.22 124.84 ± 2.01 230.90 ± 2.24 After cell disruption VEGF (pg / ml) 110.01 ± 3.52 350.07 ± 2.68 721.04 ± 4.27 1902.35 ± 3.59 After cell disruption KGF (pg / ml) 9.37 ± 0.25 55.63 ± 0.47 105.97 ± 0.49 460.04 ± 0.52 After cell disruption Procollagen (ng / ml) 83.10 ± 1.43 261.26 ± 4.61 451.10 ± 2.08 1498.66 ± 16.42

As shown in Table 3, it can be confirmed that the detection amount of the active ingredient of the cell is high as a result of Example 6. In addition, it can be expected that the detected amount will be higher than those of Example 1 and Comparative Example 1 in the case of Examples 3 to 10 that the yield is similar or larger than that of Example 1.

Hereinafter, the test for confirming the anti-arthritis and cartilage regeneration effects by the action of stem cell crush extract on chondrocytes will be described in detail.

1-1. Articular chondrocyte  Isolation and Cultivation

Before the test, the cartilage of Wistar rat female rats was recollected, cultured in DMEM medium containing 10% FBS, and confirmed to be chondrocytes using CD44, a specific marker of chondrocytes (cultivation conditions 37 ° 5% CO ² ).

1-2. result

CD44, a specific marker of chondrocytes, was used to identify chondrocytes.

2-1. Cytotoxicity and NO generation  Inhibitory effect

1) Stem cell crushing component extracts are treated in chondrocytes for 24 hours incubation and cell viability to determine the toxicity of the material to the cells.

2) Treatment of chondrocytes with IL-1α causes inflammation.

3) Inhibit the production of inflammatory mediators by inhibiting stem cell crushed extracts by concentrations on inflamed chondrocytes (* NO; Nitric Oxide).

2-2. result

1) As a result of measuring the cell viability by treatment by concentration, it did not show cytotoxicity at the concentration up to 20 ng. (Concentration 0,5,10,20 ng / ml)

2) It was confirmed that NO was increased in the cells treated with the inflammatory inducer IL-1α. Stem cell crushing extracts were treated on inflammation-induced chondrocytes and confirmed an inhibitory response of about 30% at a concentration of 20 ng / ml. )

3-1. iNOS , COX-2 and NF - kB  Change in expression

1) The correlation between NO production and COX-2 and NF-kB expression was confirmed by applying carprofen (nonsteroidal substance) and stem cell crush extracts, which are positive controls to IL-1α-treated chondrocytes.

NO: increases when inflammation occurs

COX-2: A type of protein, a substance whose activity increases due to inflammatory factors and then decreases as inflammation disappears.

2) To determine the effect of stem cell disruption extracts on the expression of NF-κB in inflamed chondrocytes.

3-2. result

1) Results of Stem Cell Fracture Extracts Treated on IL-1α-induced Chondrocytes

The inhibition of NO production induced by IL-1α treatment is associated with the inhibition of the expression of iNOS and COX-2.

2) Chondrocytes treated with stem cell disruption extracts block the NF-κB pathway, a major inflammatory signaling pathway induced by IL-1α, by inhibiting the migration of IL-1α-induced NF-κB proteins into the nucleus. It was confirmed that it can be done.

4. iMAPKs  Effect on activity

1) Treating chondrocytes with inflammation induced by IL-1α Check iMAPKs activity.

1) Confirmation of anti-inflammatory mechanism

The anti-inflammatory effect of stem cell disruption component extract is achieved through the inhibition of p38 MAPK signaling.

5-1. Factors involved in cartilage formation and development qPCR  Measure

1) Check the anti-inflammatory effect of this stem cell extract through genetic expression.

2) Confirm the cartilage formation through the gene of sox-9 which is involved in the development of cartilage formation.

5-2. result

1) MMPs are major proteins involved in catabolism of cartilage tissues, and the expression of MMP-13 was significantly increased when IL-1α was treated, but the expression was decreased when stem cell lysate extracts were treated. It confirmed that it became.

2) It was confirmed that SOX-9 genes involved in cartilage formation and development also increased gene expression according to the concentration-specific treatment of stem cell disruption extracts.

Hereinafter, the test for confirming the anti-inflammatory effect of the stem cell lysate extract will be described in detail.

1-1. Cytotoxicity Assessment

1) Stem cell disrupted extract (T-stem) was treated with mouse macrophages (RAW 246.7) at a concentration of 0.1 μg / ml to 3 μg / ml for 24 hours.

2) Simultaneously treated with stem cell lysate extract and LPS.

1-2. result

1) As shown in Figure 10, the results of treatment by concentration with the stem cell lysate extract alone, there was no statistically significant cytotoxicity.

2) As shown in Figure 11, when treated with stem cell lysate extract with 1 ㎍ / ml of Lipopolysaccharide (LPS) which is an inflammatory response inducer of mouse macrophages, there was no cytotoxicity.

2-1. Cell morphology

Stem cell lysate extract was treated with mouse macrophage (RAW 246.7) for 24 hours to 2 ㎍ / ml, the cell morphological changes were confirmed.

2-2. result

It was confirmed that the macrophage activation was relatively increased in the group treated with 1 g / ml of LPS, an inflammation-inducing factor, compared with the control group.

As shown in FIG. 12, in the cells treated with the stem cell lysate extract and 1 g / ml of LPS, the activation of macrophages occurred relatively less than the group treated with 1 g / ml of LPS alone.

3-1. Nitric oxide

Mouse macrophage cells (RAW 246.7) pretreated with LPS, a production-inducing factor, were treated for 24 hours at 0.4 µg / ml to 2 µg / ml, and then griess assay. Nitric oxide concentration was confirmed.

3-2. result

The production of nitric oxide was increased by LPS stimulation.

As shown in FIG. 13, the concentration of nitric oxide was statistically significant in the group treated with LPS and the stem cell lysate extract compared to the group treated with only LPS.

4-1. iNOS  And COX-2 protein expression changes

Mouse macrophage cells (RAW 246.7) pretreated with 1 μg / ml of LPS, an inflammation-inducing factor, were treated with test substance stem cell lysate extracts at concentrations of 1 μg / ml and 2 μg / ml for 24 hours, followed by western blot analysis. Inflammatory enzymes iNOS and COX2 protein expression was confirmed.

4-2. result

As shown in FIG. 14, it was confirmed that iNOS and COX-2 protein expression was reduced in a concentration-dependent manner in the group treated with the test substance T-STEM as compared to the LPS-treated group.

5-1. Inflammatory cytokine production

Mouse macrophage cells (RAW 246.7) pretreated with 1 μg / ml of LPS, an inflammation-inducing factor, were treated with stem cell disrupted extracts at concentrations of 1 μg / ml and 2 μg / ml for 24 hours, followed by inflammatory cytokines by ELISA assay. Production (TNFα, IL-1β) was confirmed.

5-2. result

It was confirmed that the production of inflammatory cytokines was increased by LPS stimulation.

As shown in FIG. 15 and FIG. 16, the reduction of inflammatory cytokines was observed by treatment of the test substance stem cell lysate extract.

Efficacy of the active ingredient of the stem cell crush extract according to the present invention is as follows.

First, the cause of dementia is caused by the degeneration of beta-amyloid toxic protein and tau protein.When repeated transplantation of cord blood-derived mesenchymal stem cells into both hippocampus of Alzheimer's disease mice improves memory and reduces the amount of beta amyloid in the brain. It is confirmed that the amount of β-secretase1, an enzyme that produces beta amyloid in brain tissues of mice, is decreased, the secretion of inflammatory cytokines of microglia is suppressed, and the secretion of anti-inflammatory cytokines is increased. These results also inhibit hyperphosphorylation of tau protein.

Therefore, mesenchymal stem cells are Alzheimer's disease, which inhibits the progression of dementia and reduces the amount of beta-amyloid toxic protein that causes dementia. The removed stem cell active ingredient is a dense substance that can be used to treat dementia. In addition, since the cell yield through the present invention, the yield of the active ingredient is increased, it can be used as a less effective and effective therapeutic agent than using the existing stem cells themselves.

In addition, the gum disease is caused by inflammation of the tooth roots and bones of the gums, gums, etc., the stem cells of the present invention can help stem cells to regenerate the gum, gum bone crushed extract.

That is, stem cell growth factors can be used as a gum treatment because they help promote inflammation, bone, cartilage, gum cell regeneration, and wound healing. Especially, collagen networks that look like procollagen and reticulum keep the cells firmly connected to each other. It can be used as a treatment for gum disease because it provides adhesion and provides an environment that helps many parts of the human body such as blood vessels, bones and joints to function. In addition, since the cell yield through the present invention, the yield of the active ingredient is increased, it can be used as a less effective and effective therapeutic agent than using the existing stem cells themselves.

In addition, joints and periarthritis are caused by inflammation around the joints, such as aging, injury, stem cell crush extract of the present invention can be used as a gum treatment because it helps to inhibit inflammation, bone, cartilage, gum cell regeneration, wound healing regeneration have. The collagen network, which looks like a procollagen network, firmly bonds cells to each other and provides an environment that helps cells form and function in many parts of the body, including blood vessels, bones, and joints. It can be used as a therapeutic for. In addition, since the cell yield through the present invention, the yield of the active ingredient is increased, it can be used as a less effective and effective therapeutic agent than using the existing stem cells themselves.

In addition, the causes of hair loss are various environmental factors, genetic factors, etc., VEGF growth factor of the stem cell crush extract according to the present invention helps hair follicle growth, stimulates KGF and hair roots to help strengthen hair, It can be used as a therapeutic agent. In addition, since the cell yield through the present invention, the yield of the active ingredient is increased, it can be used as a less effective and effective therapeutic agent than using the existing stem cells themselves.

In addition, the causes of skin aging are various, such as environmental factors, genetic factors, KGF, new cell production (collagen) function of the stem cell crush extract of the present invention, wrinkle prevention, recovery, skin protection, young skin maintenance and UV stimulation defense It can be used as a skin treatment because it helps the skin and collagen network to adhere firmly to each other and helps skin elasticity. In addition, since the cell yield through the present invention, the yield of the active ingredient is increased, it can be used as a less effective and effective therapeutic agent than using the existing stem cells themselves.

Atopic dermatitis composition according to the present invention comprises a stem cell crush extract and wormwood extract.

The stem cell crush extract is applied to the extract extracted by the above-described process. And wormwood extract is provided. The mugwort extract may be extracted by various methods, and in the present invention, it is preferable to extract by hot water extraction method.

It is preferable to mix 20 parts by weight of the mugwort extract to 80 parts by weight of the stem cell crush extract. This is because when the mugwort extract is 10 parts by weight or less, or 30 parts by weight or more with respect to 80 parts by weight of the stem cell crush extract.

Hereinafter, the atopic dermal composition using the stem cell lysate extract and mugwort extract according to the present invention will be described in detail through an experiment that affects the atopic skin.

Example 13

Only stem cell crush extracts were treated alone.

Example 14

Stem cell crush extract after the stock solution, wild wormwood extract was treated.

Example 15

50 parts by weight of the stem cell crush extract and 50 parts by weight of mugwort extract were mixed and treated.

Example 16

60 parts by weight of the stem cell crush extract and 40 parts by weight of mugwort extract were mixed and treated.

Example 17

70 parts by weight of the stem cell crush extract and 30 parts by weight of mugwort extract were mixed and treated.

Example 18

80 parts by weight of the stem cell lysate extract and 20 parts by weight of mugwort extract were mixed and treated.

Example 19

90 parts by weight of the stem cell lysate extract and 10 parts by weight of mugwort extract were mixed and treated.

Experimental group Skin Moisturizing (%) 0 (hr) 1 (hr) 3 (hr) a. Stem cell crushed extract applied alone. 28 35 36 b. (Stem cell extract + wild mugwort extract) application of the mixture. 30 38 44 c. After applying stem cell crushed extract, wild wormwood extract applied. 30 33 39 Experimental group Skin Moisturizing (%) 0 (hr) 1 (hr) 3 (hr) Example 13 28 35 36 Example 14 30 33 39 Example 15 26 32 36 Example 16 27 33 37 Example 17 28 35 38 Example 18 30 38 44 Example 19 30 36 37

Table 4 shows that when the first application as a result of evaluating the skin moisture level, Example 14, Example 18 and Example 19 are similar, but Example 18 is excellent over time.

Experimental group FGF-2 (pg / ml) a. Stem cell crushed extract applied alone. 310 b. (Stem cell extract + wild mugwort extract) application of the mixture. 560 c. After applying stem cell crushed extract, wild wormwood extract applied. 500 Experimental group FGF-2 (pg / ml) Example 13 310 Example 14 500 Example 15 490 Example 16 510 Example 17 530 Example 18 560 Example 19 540

As shown in Table 5, it was confirmed that Example 18 is the best for the skin regeneration factor (FGF-2).

Experimental group VEGF (pg / ml) a. Stem cell crushed extract applied alone. 160 b. (Stem cell extract + wild mugwort extract) application of the mixture. 230 c. After applying stem cell crushed extract, wild wormwood extract applied. 170 Experimental group VEGF (pg / ml) Example 13 160 Example 14 170 Example 15 170 Example 16 180 Example 17 200 Example 18 230 Example 19 220

As shown in Table 6, it was confirmed that Example 18 is the best for the skin regeneration factor (VEGF).

Experimental group TIMP-1 (ng / ml) a. Stem cell crushed extract applied alone. 1400 b. (Stem cell extract + wild mugwort extract) application of the mixture. 2000 c. After applying stem cell crushed extract, wild wormwood extract applied. 1600 Experimental group TIMP-1 (ng / ml) Example 13 1400 Example 14 1600 Example 15 1600 Example 16 1700 Example 17 1800 Example 18 2000 Example 19 2000

As shown in Table 7, it was confirmed that Example 18 is the best for the skin regeneration factor (TIMP-1).

SOAIdx Code Number SOAIdx ewe4 week 37 42 40 39 39 35 38

As shown in Table 8, it was confirmed that Example 18 was the best in the evaluation of the scale (Atopic dermatitis severity index = Chorad index) to objectively evaluate the severity of atopic dermatitis.

As such, the technical configuration of the present invention described above will be understood by those skilled in the art that the present invention can be implemented in other specific forms without changing the technical spirit or essential features of the present invention.

Therefore, the above-described embodiments are to be understood in all respects as illustrative and not restrictive, and the scope of the present invention is shown in the following claims rather than the detailed description, and the meaning and scope of the claims and their equivalents. All changes or modifications derived from the concept should be construed as being included in the scope of the present invention.

10: body portion 12: first container portion
14: 2nd container part 16: crushing part
18: valve 20: pump

Claims (3)

It comprises a stem cell crush extract and mugwort extract,
80 parts by weight of the stem cell lysate extract is mixed with 20 parts by weight of the wormwood extract is applied to the skin,
The stem cell crush extract
Cultured in a medium for mass medium,
The badge,
Basic medium;
Hyaluronic acid; And
Consisting of additive composition,
The additive composition,
Glycine, histidine, isoleucine, methionine, phenylalanine, proline, hydroxyproline, serine, threonine, tryptophan, tyrosine, valine, bFGF, EGF, VEGF, KGF, HGF, TGF, vitamin C, vitamin B1, vitamin B12, vitamin E, Consists of selenium, transferrin,
The additive composition,
The hyaluronic acid is contained in a concentration of 10 ㎍ / ㎖ for the medium composition,
The glycine is included at a concentration of 1 ng / ml,
The histidine is contained at a concentration of 1 ng / ml,
The isoleucine is contained at a concentration of 1 ng / ml,
The methionine is included at a concentration of 1 ng / ml,
The phenylalanine is contained in a concentration of 1ng / ㎖,
The proline is included at a concentration of 10 ng / ml,
The hydroxyproline is contained at a concentration of 5ng / ml,
The serine is included at a concentration of 1 ng / ml,
The threonine is included at a concentration of 1 ng / ml,
The tryptophan is included at a concentration of 1 ng / ml,
The tyrosine is contained in a concentration of 1ng / ㎖,
The valine is included at a concentration of 2 ng / ml,
The bFGF is included at a concentration of 9 μg / ml,
The EGF is included at a concentration of 1.5 μg / ml,
The VEGF is included at a concentration of 1 μg / ml,
The KGF is included at a concentration of 1.2 μg / ml,
The HGF is included at a concentration of 0.5 μg / ml,
The TGF is included at a concentration of 0.5 μg / ml,
The vitamin C is contained at a concentration of 2 μg / ml,
The vitamin B1 is included at a concentration of 0.5 μg / ml,
The vitamin B12 is included at a concentration of 3 μg / ml,
The vitamin E is included at a concentration of 500 μg / ml,
The selenium is included at a concentration of 1.8 μg / ml,
The transferrin is included at a concentration of 12 μg / ml,
The additive composition with respect to 100 parts by weight of the basic medium composition for atopic skin improvement using stem cell crushed extract and mugwort extract, characterized in that the mixture at a ratio of 5 to 40 parts by weight. delete Cosmetics prepared from the composition of claim 1.

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