KR20130047167A - Cosmetic composition containing immunocytes activation media for anti-aging - Google Patents

Abstract

PURPOSE: A method for manufacturing a cosmetic composition containing a culture medium for proliferation of immunocytes is provided to improve anti-aging and anti-wrinkling. CONSTITUTION: A method for manufacturing a cosmetic composition containing a culture medium for proliferating of immunocytes comprises: a step of preparing a medium for proliferation of immunocytes(T cells)(S11); a step of isolating lymphocytes from animal peripheral blood(S12); a step of mixing the lymphocytes in the medium and culturing the immuocytes(T cells) to prepare a mixed culture solution for proliferation of immunocytes(S13); a step of isolating the immunocytes from the mixed culture solution and preparing a culture solution for proliferation of immunocytes(S14); and a step of mixing a cosmetic ingredient with the culture solution for proliferation of immunocytes(S15). [Reference numerals] (S11) Step of preparing an adequate medium for proliferation of immunocytes(T cells); (S12) Step of extracting lymphocytes from animal peripheral blood; (S13) Step of manufacturing a mixed culture solution for proliferation of immunocytes by mixing the lymphocytes in the medium and culturing the immunocytes(T cells) included in the lymphocytes; (S14) Step of manufacturing a culture solution for proliferation of immunocytes by isolating the immunocytes from the mixed culture solution for proliferation of immunocytes; (S15) Step of mixing a cosmetic ingredient with the culture solution for proliferation of immunocytes

Description

Cosmetic composition containing immune cell proliferation culture {Cosmetic Composition containing Immunocytes activation media for Anti-aging}

The present invention relates to a method for preparing a cosmetic composition for improving skin aging using immune cell proliferation culture.

An important function of the skin is to protect the body from external stimuli such as ultraviolet rays, dry air-conditioning environment and air pollution, and play an important role in performing various roles such as thermoregulation, respiration and excretion.

As you age, these functions gradually weaken and your skin ages. In particular, as the lipid composition and content of the lipid layer which functions as a skin barrier changes, the function decreases rapidly and the skin moisture content decreases, causing the skin to dry, causing blemishes, freckles, pigmentation and various skin lesions. In addition, when the skin is exposed to strong ultraviolet rays, stress, and malnutrition, the skin accelerates aging of the skin such as wrinkle formation, loss of elasticity and keratinization.

The skin of the human body is divided into epidermis and dermis, and newly formed keratinocytes in the basal layer at the bottom of the epidermis gradually move from the lower layer to the upper layer, turning into keratin after about 14 days, and then completely aging after 14 days. Sludge or time will come off. The dermis, which is connected to the base layer, serves to hydrate the epidermis and maintain the elasticity of the skin.

Generally, skin aging such as decreased skin elasticity, wrinkles, etc. are caused by destruction of collagen, decrease in collagen synthesis, loss of elastin, etc., but may also occur in the dermis, but rather in the dermis.

In recent years, many skin physiological studies have been conducted on the causes of wrinkles and how to improve the skin. Actually, in the cosmetics, sunscreens, antioxidants, and cell activation promoters are used to prevent skin wrinkles. Wrinkle improvement methods have been proposed.

On the other hand, based on the development of biochemistry and molecular biology, a small amount of signal substances such as growth factors present in our bodies have been found, and the aging theory of living organisms is being re-established. It is also found that this signal decreases in vivo with age, and it is revealed that this growth factor is closely related to our aging. These growth factors are produced in large quantities in the stem cells that exist at the base of each tissue.

Our body is composed of about 210 cells, each of which is in charge of life activities while maintaining its own characteristics. As every living thing is alive, each cell in our body has a lifespan and dies constantly and instead is filled with new cells. It is stem cells that make this possible.

Stem cells are undifferentiated cells between differentiated cells of tissues or organs. They have self-renewing ability that continuously proliferates itself and have the ability to differentiate into all tissue cells in the body (Differentiation to specific tissue). Pluripotent cells. Adult stem cells include hematopoietic stem cells, mesenchymal stem cells, neuroblasts, epithelial hair cells, etc., and hematopoietic stem cells can produce blood cells and lymphocytes, and are useful for treating immune system diseases including blood cancer. In case of differentiation into bone, cartilage, adipose and fibrous tissues are involved in the recovery of each tissue is damaged. However, these adult stem cells are present in small amounts in their tissues and may remain silent for years without dividing or proliferating. That is, stem cells are 'undifferentiated' cells that have such differentiation ability but have not yet differentiated. Under these undifferentiated conditions, if appropriate conditions are met, stem cells can differentiate into various tissue cells.

Stem cells are also largely divided into embryonic stem cells and adult stem cells. Stem cells can be divided into two types: adult stem cells, which are present in various tissues in our bodies since birth, and embryonic stem cells, which are derived from fertilized eggs, which are the beginning of human life. . Adult stem cells are cells that constitute specific tissues, that is, bone marrow cells are blood cells, skin stem cells are skin, and neurons are destined to differentiate only olfactory nerve cells. Which cells differentiate into stem cells depends on the environment in which they are cultured.

Human adipocyte conditioned media extracts (Human adipocyte conditioned media extracts), which are widely used in cosmetic compositions, are used to culture stem cells from adipose tissue for several generations to separate stem cells, and then to produce stem cells from stem cell culture. You can get the protein. This protein mixture contains more than 150 growth factors including vEGF (vascular endothelial growth factor), TGF (transformation growth factor), HGF (hepatocyte growth factor), FGF (fibroblast growth factor), and IGF (insulin-like growth factor). growth factor) is reported to contain a protein component. Adipose stem cells are a type of adult stem cells that are easier to harvest than bone marrow stem cells or umbilical cord blood stem cells.

The growth factor is the most important factor in the proliferation and differentiation of one cell into another. This growth factor plays a role in transmitting signals that cause cells to proliferate or differentiate into cells, and based on these signals, cells divide and differentiate to make the skin fresh and lively and always keep the skin fresh and fresh. You can keep it in the best condition. When the proteins produced from stem cells were treated with fibroblasts of the skin, the amount of collagen, a connective tissue produced by fibroblasts, increased by five times or more, and the proliferation of fibroblasts increased by more than 30%. Fibroblasts are cells that produce collagen, which forms the dermal layer of the skin, and when the amount of collagen decreases, skin elasticity decreases and wrinkles increase.

Stem cells are multipotent and multipotent cells that have the ability to differentiate into cells such as bone, cartilage, ligaments, fat, myocardium, and muscle in vitro as well as in vitro. . These multipotent cells, stem cells, are not only used as a good model in the study of differentiation mechanisms of individual tissue cells, but are also used in the development of various cell therapies.

The use of stem cells in the field of cosmetic compositions uses growth factors as their metabolites. Growth factors are proteins that bind to receptors on the cell's surface and stimulate cell proliferation and differentiation. Functions of growth factors are so diverse that they stimulate the division of many cells, while in some cases only specific cell types. Growth factors have been shown to regulate cell differentiation, migration and proliferation as essential components for stimulation of cell proliferation. Binding of growth factors to specific receptors activates signal transduction chains, which transmit signals from the outside of the cell to the inner nucleus. As signal transduction continues, it acts like a large amount of other transporters, finally synthesizing new proteins. Thus, growth factors form important signal transduction networks in the transmission of intercellular interactions and in the control of function. In addition, skin wound regeneration experiments in mice showed that the wound size was reduced by more than 40% when the stem cell-derived protein was not applied. In addition, it shows good results in the whitening effect for pigmented humans, ensuring strong efficacy and safety as a raw material for cosmetic composition.

Therefore, if you create an environment to differentiate into neurons becomes a neuron, and if you create an environment to differentiate into skin cells can be a skin cell. Research for applying the stem cell culture extract to the cosmetic composition composition using these properties is in progress.

Korean Patent Application No. 2005-55017 discloses wrinkle improvement and / or containing allogeneic and / or autologous adipocytes, stem cells isolated from allogeneic and / or autologous adipocytes, or fibroblast growth factors isolated therefrom. The anti-aging cosmetic composition is known, the Korean Patent Registration No. 848056 is known a whitening cosmetic composition comprising a stem cell culture medium or a protein isolated from the culture solution, the Republic of Korea Patent No. 0735081 invention is CD4 Methods for activating T cells are known, and Korean Patent Application No. 2007-15211 contains a conditional medium obtained by culturing human adipose stem cells in serum-free medium containing antioxidants, vitamins, amino acids and minerals for at least one day. Injectable additives for use in human stem cell-containing injectables for tissue regeneration are known. .

   However, such human-derived stem cells have not been secured, and thus still have limitations in safe use in cosmetic compositions, and particularly ethical problems remain, which have many limitations for their use in the future. Difficult to penetrate the skin of a variety of active ingredients, more specifically, a variety of cell growth factors, which has a good effect on medical treatment purposes, unlike cosmetics have a limited effect on normal skin. Therefore, in order to improve the skin aging phenomenon, dermatology is directly injecting stem cell culture extract derived from human body into the skin or inducing skin penetration by using a special device to induce skin penetration. There is a problem of using this method as a cosmetic is difficult.

The present invention is to provide a method for producing a cosmetic composition containing an immune cell proliferation culture.

The present invention is to provide a method for producing a cosmetic composition containing the immune cell proliferation culture is effective in skin aging and wrinkle improvement.

According to one aspect of the invention,

(a) forming a medium suitable for immune cell (T cell) proliferation;

(b) extracting lymphocytes from peripheral blood of the animal;

(c) preparing an immune cell proliferation culture mixture by culturing the lymphocytes in the medium and culturing immune cells (T cells) contained in the lymphocytes;

(d) separating the immune cells from the immune cell proliferation culture mixture to prepare an immune cell proliferation culture solution;

(e) Provided is a method for producing a cosmetic composition containing an immune cell proliferation culture medium comprising the step of mixing the cosmetic component in the immune cell proliferation culture medium.

In addition, the step (b),

Peripheral blood was superimposed on a 1.077 specific gravity Ficoll-Paque Plus solution and centrifugally precipitated, and red blood cells and granulocyte layers with specific gravity greater than 1.077 were down, and mononuclear cell layers were 1.077 or less, depending on the difference in specific gravity. Hyperplatelets and the like are separated to obtain a mononuclear cell layer containing lymphocytes, and a method for producing a cosmetic composition containing an immune cell proliferation culture solution, comprising the steps of extracting only lymphocytes from the isolated mononuclear cell layer.

In addition, the step (c),

(c1) The lymphocytes harvested through step (b) are cultured in the presence of anti-CD28 and anti-CD16 in the culture medium to which L-glutamine and plasma are added, thereby increasing the primary immune cell proliferation culture mixture. Preparing a;

(c2) Plasma is additionally added to the primary immune cell proliferation culture mixture, and the primary immune cell proliferation culture mixture is injected into the gas permeable culture bag containing the culture solution, followed by further culturing to increase the lymphocytes in large quantities. Provided is a method for preparing a cosmetic composition containing an immune cell proliferation medium comprising the steps of preparing an immune cell proliferation medium.

Also, in the step (d)

(d1) centrifuging to separate the immune cells and the immune cell proliferation culture into the upper and lower layers, respectively;

(D2) there is provided a method for producing a cosmetic composition containing the immune cell proliferation culture medium comprising the step of filtering the separated immune cell proliferation medium using a cell strainer (Cell Strainer) and a micro filter.

The present invention provides a method for producing a cosmetic composition containing the immune cell proliferation culture.

The present invention provides a method for producing a cosmetic composition containing the immune cell proliferation culture is effective in skin aging and wrinkle improvement.

1 is a flow chart of a method for producing a cosmetic composition containing an immune cell proliferation culture medium according to an embodiment of the present invention.

The present invention is an immune cell proliferation culture mixture having excellent effect on skin beauty by activating and culturing T cells (immune cells) using anti-CD28 (anti-CD28) antibody as an additive in cultured lymphocytes derived from peripheral blood of animals. Where the immune cell proliferation culture is separated from the immune cell proliferation culture mixture.

Prior to the detailed description of the present invention, first, with the characteristics of T cells (immune cells), the mechanism of action of each of the additives used for immune cell amplification and activation will be described.

T cells refer to cells having a T cell antigen receptor (TCR) on the cell surface. TCR forms a heterodimer with the CD3 antigen of the di- and β-chain dimers. Some of the T cells (around 5% of peripheral blood T cells) are dimers of γ and δ chains, not αβ. TCRs form complexes with CD3 antigens (γ, δ, ε, ζ, ζ or η). CD3 antigens are responsible for delivering signals within cells when TCR recognizes antigens.

In general, helper T cells that promote an immune response to cancer cells release various cytokines to activate killer T cells, B cells, and macrophages. The types of cytokines are interleukin, an information carrier between cells. There are various substances such as 1, 2, 3, TNF-α and interferon-γ. In the present invention, anti-CD28 (anti-CD28) antibody and anti-CD16 (anti-CD16) antibody are added to the culture medium to activate immune cells such as killer T cells, B cells, and macrophages.

IL-2 is a glycoprotein with a molecular weight of 14 to 17 kDa that is produced when T cells recognize and activate an antigen.It is secreted out of T cells and then reacts with the T cells that produce IL-2 itself. Promotes cell growth. IL-2 also acts on NK cells to promote growth, enhances NK cell killing capacity, and acts on B cells to promote their growth.

NKT cells are a type of T cell whose function is being revealed relatively recently as an attendant of endogenous immunity. As the name suggests, it expresses T-cell receptors and NK cell-specific surface markers. One surprising feature of NKT cells is the rapid release of several cytokines such as IL-4, IL-10, IL-13, IFN-γ, TNF-α after activation. This feature suggests that NKT cells can have a significant effect on the adaptive immune response.

Therefore, the present invention uses anti-CD28 monoantibody, anti-CD16 monoantibody in culture to activate immune cells such as T cells from lymphocytes of peripheral blood of animals.

The present invention is intended to be described more clearly by the following examples, experimental examples and comparative examples, but the present invention is not intended to be limited thereto.

Hereinafter, a method for preparing immune cell proliferation culture medium according to the present invention will be described in detail. The "immune cell proliferation culture liquid" referred to in the present invention is a mixture of immune cells and immune cell proliferation culture solution. Immune cell proliferation culture can be obtained by isolating immune cells from the immune cell proliferation culture mixture. Immune cell proliferation broth is a mixture of secondary metabolites of immune cells.

In the present invention, after proliferating immune cells (T cells), the immune cell proliferation culture mixed with secondary metabolites produced therefrom is utilized as a cosmetic ingredient.

Therefore, the present invention can be broadly divided into the process of proliferation of immune cells and the process of preparing a cosmetic composition.

1 is a flow chart of a method for producing a cosmetic composition containing an immune cell proliferation culture medium according to an embodiment of the present invention. S14 to S14 is a method for producing an immune cell proliferation culture medium, and finally, the cosmetic composition is completed by mixing the cosmetic components in S15.

S11 is a step of forming a medium suitable for immune cell (T cell) proliferation.

For the present invention, first, a medium must be prepared in an environment in which immune cells can proliferate. First, amino acids are dissolved in purified water to obtain respective solutions. Inorganic compounds are dissolved in purified water to obtain solutions. Organic compounds are mixed in mineral solution. Each vitamin is dissolved in purified water to obtain a solution, and a mixture of lipids and proteins is obtained. Mixing these solutions in the proper proportions completes the medium.

With respect to the total weight of the medium, the inorganic solution contains 0.1 to 100% by weight, preferably 5 to 20% by weight, the amino acid solution is 0.1 to 100% by weight, preferably 0.1 to 10% by weight, and the vitamin solution is 1 to 100% by weight, preferably 0.01 to 10% by weight, protein solution is 0.1 to 100% by weight, preferably 1 to 10% by weight, lipid solution is 0.1 to 100% by weight, preferably 0.01 to 2% by weight. The composition of this medium may be as shown in Table 1.

ingredient Unit: weight% Human Oligopeptide-1,2 0.01
A


















L-Alanine 0.2 L-Asparagine 0.06 L-Arginine 0.06 L-Aspartic acid 0.02 L-Cystine 0.04 L-Glutamic acid 0.02 L-Glutamine 0.7 L-Glycine 0.01 L-Histidine 0.02 L-Isoleucine 0.05 L-Leucine 0.05 L-Lysine 0.04 L-Methionine 0.02 L-Phenylalanine 0.02 L-Proline 0.02 L-Serine 0.03 L-Threonine 0.02 L-Tryptophan 0.01 L-Tyrosine 0.02 L-Valine 0.02 Taurine 0.1

B




CaCl ₂ 0.05 MgSO ₄ 0.05 KCl 0.4 NaCl 6.0 NaH ₂ PO ₄ 0.8 KNO ₃ 0.05 Na ₂ SeO ₃ 0.00001 NaHCO ₃ 2.0
C

2-amino ethanol 0.001 D-Glucose 2.0 HEPES 2.0 Sodium pyruvate 0.11

D









D-Biotin 0.0002 Cyanocobalamin 0.000005 Cholin chloride 0.003 Folic acid 0.001 myo-Inositol 0.035 Niacinamide 0.001 D-1 / 2 Ca Pantothenate 0.00025 Pyridoxine 0.001 Thiamine 0.001 Cholecalciferol 0.002 Ergocalciferol 0.002 Ascorbic acid 0.002 Riboflavin 0.0002 E
Linoleic acid 0.015 Oleic acid 0.015
F Apotransferrin 0.005 Albumin 2.0 Purified water Balance system
100

S12 is the step of extracting lymphocytes from the peripheral blood of the animal.

Mononuclear cells, such as lymphocytes or monocytes of the animal, have a specific gravity lower than 1.077, and the peripheral blood of the animal is superimposed on 1.077 specific gravity Ficoll-Paque Plus solution and centrifuged by constant centrifugal force. According to the difference, red blood cells and granulocyte layers having a specific gravity greater than 1.077 are separated, and mononuclear cell layers and platelets, which are 1.077 or less, are separated upwards to obtain mononuclear cell layers including lymphocytes, and only lymphocytes are extracted from the separated mononuclear cell layers. This lymphocyte extract contains immune cells (T cells).

S13 is a step of preparing an immune cell proliferation culture mixture by mixing the lymphocytes with the medium to proliferate (culture) the immune cells (T cells) contained in the lymphocytes.

The culture step may be divided into a primary culture step and a secondary culture step.

1. Primary culture step-Preparation process of primary immune cell proliferation culture mixture

Lymphocytes harvested through the lymphocyte extraction step (S12) are incubated for 6-8 days in the presence of anti-CD28 and anti-CD16 in a culture medium to which L-glutamine (L-glutamine) and plasma are added. The specific process is as follows.

First, the harvested lymphocytes were suspended in 3 ml of culture medium, and then, 20-100 µl of L-glutamine, 1-2 ml of plasma, 1-10 µl of anti-CD16, and 25-45 ml of culture medium were solidified. Incorporate into a 75 cm 2 culture vessel, and incubate for 3-4 days in a 37 ℃ CO ₂ incubator. Through this procedure, a primary immune cell proliferation culture mixture is prepared.

2. Secondary Culture Stage-Secondary Immune Cell Proliferation Culture Mixture Preparation Process

Plasma (3-5 ml) is added to the immune cell proliferation culture mixture (29-49 ml) that has undergone the first culture step, and the newly added immune cell proliferation culture mixture is 1 liter of gas permeable culture bag. After injecting into the cells, lymphocytes are grown in large amounts by further incubating for 3 to 4 days.

Through the first and second culture stages as described above, the number of T cells (immune cells) increases in mass and the size of each T cell also increases. As a result of the actual culture experiments, the total lymphocyte count when separating lymphocytes from 30cc blood was 1.0x10. ^{From 6} to 3.0x10 ⁷ was finally increased to 1.0x10 ⁹ ~ 5.0x10 ^9. This process produces a secondary immune cell proliferation culture mixture.

In step S14, the immune cells are separated from the immune cell proliferation culture mixture to prepare an immune cell proliferation culture solution. Step S14 may be divided into centrifugation to separate the immune cells and immune cell proliferation culture into the upper and lower layers, respectively, and filtering the separated immune cell proliferation culture using a cell strainer and a filter. .

1. Centrifugation to separate the immune cell and immune cell proliferation medium into the upper and lower layers, respectively

The immune cell growth culture mixture incubated in a 1000 mL gas permeable culture bag was transferred to a 500 mL sterilized centrifuge tube and centrifuged at 1000 rpm to 2500 rpm for 15 minutes to 25 minutes to obtain immune cells (lower layer solution) and immune cell growth culture medium ( Supernatant).

2. Filtering the Isolated Immune Cell Proliferation Culture Cell Using a Cell Strainer and a Filter

The isolated immune cell proliferation culture medium is completely separated from the immune cells and immune cell proliferation medium using a cell strainer (Cell Strainer, 70 μm pore size) and a micro filter (0.2 μm pore size), and then filled into a sterile container.

This step S14 virtually separates the entire lymphocytes, including T cells.

Through the above steps S11 and S14 to prepare an immune cell proliferation culture. Hereinafter, a method for preparing a cosmetic composition using immune cell proliferation culture will be described.

Cosmetic compositions can be completed by combining existing cosmetic ingredients, in accordance with various purposes and properties. The cosmetic composition of the present embodiment is only one embodiment, and the immune cell proliferation culture medium of the present invention is mixed with existing cosmetic ingredients to prepare various cosmetic compositions.

≪ Example 1 > Preparation of cosmetic composition containing immune cell proliferation culture solution according to the invention

To prepare a cosmetic composition containing a mixture of immune cell proliferation culture made of the composition shown in Table 2 below.

ingredient Unit: weight% Delta-tocopherol 0.2 Immune cell proliferation culture 5.0 A Cetostearyl alcohol 2.0 Glyceryl stearate 1.5 Microcrystallin 0.7 Squalane 5.0 Liquid paraffin 3.0 Trioctanoine 5.0 Polysorbate 60 1.2 Sorbitan stearate 0.5 Cyclomethicone 3.0 Tocopheryl acetate 0.2 BHT 0.05 B Purified water Balance Concentrated glycerin 4.0 1,3-butylene glycol 2.0 Xanthan gum 0.1 EDTA-2Na 0.05 Incense, preservative Suitable amount system 100

< Comparative example  1> Human body  Preparation of cosmetic composition containing fat stem cell culture extract

The cosmetic composition of Comparative Example 1 was prepared by containing the human-derived fat stem cell culture in the remaining cosmetic composition (see Table 2) except for the immune cell proliferation culture in the composition of Table 1.

ingredient Unit: weight% Delta-tocopherol 0.2 Human-derived fat stem cell culture fluid 5.0 A Cetostearyl alcohol 2.0 Glyceryl stearate 1.5 Microcrystallin 0.7 Squalane 5.0 Liquid paraffin 3.0 Trioctanoine 5.0 Polysorbate 60 1.2 Sorbitan stearate 0.5 Cyclomethicone 3.0 Tocopheryl acetate 0.2 BHT 0.05 B Purified water Balance Concentrated glycerin 4.0 1,3-butylene glycol 2.0 Xanthan gum 0.1 EDTA-2Na 0.05 Incense, preservative Suitable amount system 100

Experimental Example 1 Skin Wrinkle Improvement

1. Instrumental evaluation

In order to measure the skin wrinkle improvement effect (mechanical evaluation) for the cosmetic composition prepared according to Example 1 and Comparative Example 1 according to the present invention, 20 women (age 28.5 years) of 20 or older (average age 28.5 years), respectively, Divided into groups, the cosmetic composition prepared in Example 2 according to the present invention was applied to group A, and the cosmetic composition prepared in Comparative Example 1 was applied to group B.

A group of A and B groups were applied to the area of the upper arm 2X2 cm 2 (2 times / day, 0.2 g / time) for 6 weeks, and a replica of the skin wrinkles was made using a transparent silicone solution. Skin wrinkles were measured by a wrinkle measuring instrument (SKIN VISIOMETER SV400, C + K Electronics GmbH. Germany). The replica image was analyzed three-dimensionally with a CCD camera, and the average wrinkle roughness (R _z ) of Equation 1 below was obtained by dividing the sum of roughness (R _m : m is an integer of 1 or more) of each wrinkle by the number of wrinkles. Wrinkle improvement effect was analyzed. The test results are shown in Table 4 below.

Skin wrinkle improvement effect (6 weeks) Average wrinkle roughness (R _z, ㎛) Example 2 Comparative Example 1 T0 T6 ΔR _z T0 T6 ΔR _z Subject 1 122 60 -62 120 86 -34 Subject 2 120 33 -87 117 42 -75 Subject 3 96 22 -74 132 60 -62 Subject 4 121 46 -75 115 59 -56 Subject 5 119 25 -94 128 50 -78 Subject 6 140 27 -113 125 33 -92 Subject 7 133 35 -98 120 49 -71 Subject 8 136 33 -103 142 108 -34 Subject 9 115 20 -95 98 16 -82 Subject 10 123 51 -72 119 44 -75 Average -87.3 -65.9

As can be seen in Table 4, Example 6 according to the present invention after 6 weeks the average wrinkle roughness (R _z ) is 87.3 ㎛ (p <0.01), Comparative Example 1 as 65.9 ㎛ (p <0.01) , Cosmetics according to the present invention was improved by about 32.5% more wrinkles than Comparative Example 1.

Therefore, the cosmetic composition containing the immune cell proliferation culture solution according to the present invention can be seen that the effect of improving wrinkles very effectively.

2. Visual Evaluation

In order to measure the skin wrinkle improvement effect (visual evaluation) for the cosmetic composition prepared in Example 1 and Comparative Example 1 according to the present invention, 20 women (age age 28.5 years) of 20 years old or older, respectively, group A and B Divided into cosmetic composition prepared in Example 2 according to the present invention was applied to the group A, the cosmetic composition prepared in Comparative Example 1 was applied to the group B.

After six weeks of application (2 times / day) around wrinkled eyes on each subject of group A and B, improvement of skin wrinkles was classified by evaluating subject's subjective evaluation and subject's subjective evaluation into the following six levels. Degree (ΔW) was measured. The results are shown in Tables 5 and 6 below.

<Evaluation criteria for skin wrinkle improvement>

-3: extremely badly -2: worsening -1: slightly worsening

0: no change 1: slightly improved 2: improved 3: highly improved

 Clinical Effects of Skin Wrinkle Improvement (Objective Evaluation) Skin wrinkle improvement (△ W) Example 2 Comparative Example 1 T0 T6 T0 T6 Subject 1 0 3 0 3 Subject 2 0 3 0 2 Subject 3 0 3 0 3 Subject 4 0 2 0 2 Subject 5 0 3 0 One Subject 6 0 3 0 3 Subject 7 0 3 0 2 Subject 8 0 3 0 3 Subject 9 0 2 0 3 Subject 10 0 2 0 2 △ W (T6-T0) 2.7 2.4

 Clinical effect of skin wrinkle improvement (subjective evaluation of subject) Skin wrinkle improvement (△ W) Example 2 Comparative Example 1 T0 T6 T0 T6 Subject 1 0 3 0 2 Subject 2 0 2 0 3 Subject 3 0 3 0 One Subject 4 0 2 0 2 Subject 5 0 3 0 2 Subject 6 0 2 0 One Subject 7 0 3 0 3 Subject 8 0 3 0 3 Subject 9 0 2 0 3 Subject 10 0 2 0 2 △ W (T6-T0) 2.5 2.2

As can be seen in Table 5 and Table 6, in Example 1 according to the present invention, the objective evaluation of the skilled person and the subjective evaluation of the subject were 2.7 and 2.5, respectively, 12.5% in the objective evaluation of the skilled person compared to Comparative Example 1 In the subjective evaluation of the increase of 13.6%, it can be seen that the cosmetics containing the immune cell proliferation culture solution of the present invention improves wrinkles more effectively than the cosmetics containing human fat stem cell culture extract.

< Experimental Example  3> Skin whitening effect: From Melanosite  Inhibitory effect on melanin production

Melanosite was used by purchasing a mouse-derived B-16 melanoma (ATCC CRL 6323) cell line. Melanoma cell lines were inoculated in DMEM medium containing 4.5 g / L glucose, 10% serum and 1% antibiotics and incubated at 37 ° C. in a 50 ml T-flask. 5% CO ₂ After culturing for 24 hours under the conditions, cells were isolated by treatment with 0.05% Trypsin containing 0.02% EDTA, and then inoculated in a 50 ml T-flask and incubated for 48 hours. At this time, the cell number is 5.76 × 10 ⁶ cells / flask. Herein, the immune cell proliferation culture solution of Example 1 and the human-derived fat stem cell culture extract used in Comparative Example 1 according to the present invention were diluted in DMEM medium and treated to cultured melanoma cells at 37 ° C. Incubate daily. After incubation, all of the medium was removed, cells were treated with 1 ml of saline-phosphate buffer (PBS) containing 0.02% EDTA and 0.05% trypsin, and then cells were collected by centrifugation for 5 minutes. 5% trichloroacetate (TCA) is treated, stirred and centrifuged to wash the precipitated melanin with saline-phosphate buffer. 1N NaOH was dissolved in the melanin precipitated, and then the absorbance was measured at 475 nm. Melanin concentrations were determined from standard concentration curves of synthetic melanin (Sigma). The experimental results are shown in Table 7.

 Inhibitory Effect of Melanin Synthesis in Melanosite density
(Μg / ml) Melanin production inhibition rate (%) Immune cell proliferation culture Human-derived stem cell culture medium No treatment - - One 6.4 4.7 2 18.8 12.3 5 23.1 20.5 10 39.9 33.3 20 61.2 55.1 50 79.4 58.9 100 107.4 65.8

As can be seen from Table 7, the immune cell proliferation culture medium of Example 1 according to the present invention exhibited a higher melanin synthesis inhibitory effect when treated with the human adipose stem cell culture medium used in Comparative Example 1, thus the present invention is melanosite. It can be seen that the skin whitening effect by inhibiting the production of melanin.

< Experimental Example  4> Skin elasticity improvement effect

The skin elasticity-improving effect of Example 2, which is a cosmetic composition containing the immune cell proliferation culture medium of the present invention, and Comparative Example 1, which is a cosmetic composition containing the human-derived fat stem cell culture extract were measured.

The cosmetic composition prepared according to Example 1 according to the present invention was divided into 20 groups (average age 28.5 years), respectively, at a temperature of 24-26 ° C. and a humidity of 45%. , The cosmetic composition prepared by Comparative Example 1 was applied to group B around the eyes for 6 weeks (2 times / day), and then using a skin elasticity measuring instrument (Cutometer SEM 575, C + K Electronic Co., Germany). Skin elasticity was measured. The test results are described in Table 7 as ΔR (R (before use) -R8 (after use)) value of Cutometer SEM 575, where R value represents the nature of skin viscoelasticity.

Skin elasticity (after 6 weeks) Skin viscoelasticity improvement degree (△ R) Example 1 Comparative Example 1 Subject 1 0.45 0.43 Subject 2 0.37 0.41 Subject 3 0.49 0.30 Subject 4 0.51 0.42 Subject 5 0.27 0.33 Subject 6 0.44 0.25 Subject 7 0.46 0.28 Subject 8 0.59 0.31 Subject 9 0.22 0.24 Subject 10 0.45 0.35 △ R 0.43 0.33

As can be seen from the results of Table 8, the cosmetic Example 1 containing the plant stem cell extract mixture was increased by 30.3% skin elasticity compared to Comparative Example 1.

< Experimental Example  5> moisturizing effect

The skin moisturizing effect of Example 1 which is a cosmetic composition containing the immune cell proliferation culture of the present invention and Comparative Example 1, which is a cosmetic composition containing a human-derived fat stem cell culture was measured. The measurement was performed after stabilizing the skin for 20 to 30 minutes in a room having a temperature of 24-26 ° C., a relative humidity of 45%, and no air flow. The cosmetic composition prepared by Example 1 according to the present invention is divided into 20 groups (average age 28.5 years) of 20 women or older, respectively, and the cosmetic composition prepared by Comparative Example 1 After 6 weeks of application (2 times / day) around the eyes of group B, TEWAMETER TM210 (C + K electronic GmbH. Germany) was used to measure transdermal moisture loss, that is, TEWL (Transepidermal Water Loss) value over time. The moisturizing effect was measured by measuring the amount of change in TEWL value ΔTEWL and using the CORNEOMETER CM 820 PC (C + K electronic GmbH. Germany) to quantify the moisture content of the skin from 0 to 150 with the change of electrical conductivity according to the epidermal moisture content. The test results are shown in Table 9 below.

 Skin moisturizing effect Test Product △ TEWL Corneometer value Example 2 5.2 119 Comparative Example 1 3.3 101

로 비교예 1에 비해 경피수분손실량이 6주 후 매우 효과적으로 개선되었음을 알 수 있다.As a result of the test, the ΔTEWL value of Example 1 was 5.2.

As compared with Comparative Example 1, it can be seen that the transdermal moisture loss was improved very effectively after 6 weeks.

In addition, in the Corneometer value measuring the moisture content of the skin, it can be seen that Example 1 is increased by 17.8%, respectively, compared to Comparative Example 1, the skin is very moist.

< Experimental Example  6> Skin Safety Test

Skin safety test was performed on the cosmetic composition containing the immune cell proliferation culture medium of Example 1 according to the present invention.

Twenty subjects (mean age 26.7 years, age 18-18 years) were subjected to a patch test on the upper arm using Haye's Test Chamber. Psoriasis, Eczema, and other skin lesion holders, or those who are pregnant, nursing or contraceptives, and antihistamines, were excluded from this study. After wiping the test site with 70% ethanol and drying, dropping the cosmetics prepared in Example 1 and Comparative Example 1 according to the present invention into 15μg each chamber, and then fixed on the upper portion of the test site. Apply the patch for 24 hours and after removing the patch, mark the test site with a marking pen and observe the test site after 24 hours and 48 hours, respectively. Determination was performed 24 hours and 48 hours after patch removal, and skin reactions were determined according to the regulations of the International Contact Dermatitis Research Group (ICDRG) shown in Table 10 below. The test results are shown in Table 10 below.

 Criteria for international contact dermatitis sign Criteria evaluation Mean Score ±
+
++
+++
- Doubtful or slight reaction and erythema
Erythema + Induration
Erythema + Induration + Vesicle
Erythema + Induration + Bullae
Negative Smile stimulation
Light stimulus
Moderate irritation
River stimulation
No stimulation 0 to 0.9
1.0-2.9
3.0-4.9
5.0 or more
0

Skin safety Elapsed time Example 2 Comparative Example 1 24 hours 0 0.0 48 hours 0 0.0

As can be seen in Table 11, as a result of the skin safety test, it can be seen that in Example 1 and Comparative Example 1 according to the present invention, the average degree of irritation is 0, which is a safe cosmetic without irritation on the skin.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Modifications and additions by those skilled in the art to an equivalent range based on the embodiments will also fall within the scope of the present invention.

none

Claims (4)

(a) forming a medium suitable for immune cell (T cell) proliferation;
(b) extracting lymphocytes from peripheral blood of the animal;
(c) preparing an immune cell proliferation culture mixture by culturing the lymphocytes in the medium and culturing immune cells (T cells) contained in the lymphocytes;
(d) separating the immune cells from the immune cell proliferation culture mixture to prepare an immune cell proliferation culture solution; And
(e) A method for preparing a cosmetic composition containing an immune cell proliferation culture medium comprising mixing a cosmetic ingredient into the immune cell proliferation culture medium.


The method of claim 1,
The step (b)
Peripheral blood of the animal was superimposed on 1.077 specific gravity Ficoll-Paque Plus solution and centrifugally precipitated, and the red blood cells and granulocyte layers having a specific gravity greater than 1.077 were down, and the mononuclear cell layer was 1.077 or less depending on the difference in specific gravity. And platelets are separated to obtain a mononuclear cell layer containing lymphocytes, and extracting only lymphocytes from the isolated mononuclear cell layer.
The method of claim 1,
The step (c)
(c1) The lymphocytes harvested through the step (b) are cultured in the presence of anti-CD28 and anti-CD16 in a culture medium to which L-glutamine and plasma are added. Preparing a;
(c2) Plasma is additionally added to the primary immune cell proliferation culture mixture, and the primary immune cell proliferation culture mixture is injected into the gas permeable culture bag containing the culture solution, followed by further culturing to increase the lymphocytes in large quantities. Method for producing a cosmetic composition containing an immune cell proliferation medium comprising the step of producing an immune cell proliferation medium.
The method of claim 1,
The step (d)
(d1) centrifuging to separate the immune cells and the immune cell proliferation culture into the upper and lower layers, respectively;
(D2) The method for producing a cosmetic composition containing the immune cell proliferation culture medium comprising the step of filtering the separated immune cell proliferation medium using a cell strainer (Cell Strainer) and a micro filter.

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