KR102043739B1 - Cosmetic Composition containing Human Adipocyte Conditioned Media Extract and Polydeoxyribonucleotide - Google Patents

Abstract

An object of the present invention is to provide a cosmetic composition containing human fat stem cell culture extract and PDRN, specifically for wrinkle improvement, whitening containing human fat stem cell culture extract and PDRN (PDRN) To provide a skin composition for enhancing skin elasticity, moisturizing and promoting hair growth.

Description

Cosmetic composition containing human adipose stem cell culture extract and PDAL {Cosmetic Composition containing Human Adipocyte Conditioned Media Extract and Polydeoxyribonucleotide}

The present invention relates to a cosmetic composition containing human fat stem cell culture extract and PDn, specifically, for improving wrinkles, whitening, elasticity, moisturizing and hair growth containing human fat stem cell culture extract and PDn It relates to a cosmetic composition for promotion.

The greatest areas of interest in cosmetics are wrinkles, pigmentation, decreased elasticity, skin drying and hair loss or lack of gloss associated with aging. The skin undergoes various changes through aging. First, the thickness of the skin's epidermis, dermis and subcutaneous tissue becomes thin and the ECM (Extracellular Matrix) component that gives elasticity to the skin changes.

Collagen, elastin, proteoglycans, glucosaminoglycan, laminin, fibronectin, etc., constituting skin connective tissue, are oxidized and function. By losing, the skin loses its elasticity and excessive wrinkles form, turning it into aged skin. In particular, collagen, which accounts for 70% to 80% of ECM, has a close relationship with wrinkle formation as its production rapidly decreases with age.

The connective tissue fibers of the ECM (extracellular matrix) include glue fibers (collagen fibers, collagen fibers), reticulated fibers, and elastic fibers (elastic fibers, elastic fibers), which account for about 70% of skin connective tissue. Collagen in most forms in the fibroblasts of the skin. As we age, collagen content in skin connective tissues decreases due to a decrease in collagen synthesis and accelerated degradation. Therefore, degradation of collagen biosynthesis and promotion of collagen degradation may be the main cause of skin wrinkle formation.

Collagen biosynthesis processes are regulated by many factors involved in transcription and post-translation levels, and collagen degradation is collagenase, which breaks down collagen by UV light. By promoting the expression of matrix metalloproteases (MMP), such as collagen degradation is promoted to reduce the collagen content. In addition, as the deformation of collagen is accelerated by the external environment, the skin becomes wrinkled and deepened. As a result, skin aging is manifested by cell homogenization, loss of elastin, destruction of collagen, reduction and delay in collagen synthesis. Thus, skin aging occurs in the skin epidermis, but rather in the dermis.

Before aging, the skin cells are physiologically active. In fibroblasts of dermis, collagen and metabolism, which are important for inhibiting skin elasticity and wrinkle formation, are active. The synthesis of glycosaminoglycans (GAGS), a glycoprotein that contains hyaluronic acid, which is essential for maintaining skin moisture, is also active, giving skin elasticity, softness, flexibility and moisturizing properties.

In the epidermis, the proliferation of basal layer cells is active, and the production of adhesion proteins such as laminin, integrin, and desmosome, which enhance the binding between skin cells, is balanced. Keeps your skin healthy. However, the skin of modern people is exposed to various harmful environments and stresses and suffers various damages. As the skin ages, the physiological activity of the skin decreases and the recovery rate of the damaged skin slows down, the skin loses elasticity, dryness, wrinkle formation and pigmentation. Various skin aging phenomena such as deposition, blotch, and dullness appear. Therefore, the main goal of the cosmetics to improve the aging symptoms, various cosmetics for this is being developed.

In general, skin aging such as decreased skin elasticity, wrinkles, pigmentation, etc. are caused by destruction of collagen, decrease in collagen synthesis, loss of elastin and melanin pigmentation due to deterioration of skin physiology. It is a phenomenon. Recently, many skin physiological researches on skin aging have been conducted. Actually, sunscreens, antioxidants, and cell activation promoters have been used to prevent skin wrinkles in cosmetics, and methods for improving skin wrinkles due to collagen synthesis have been suggested. It is becoming.

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.

It effectively protects the skin from harmful factors such as ultraviolet rays during daylight, but as the age increases, the skin tissue gradually changes, which lowers the ability to protect against daylight, resulting in wrinkles, pigmentation, sagging skin, loss of skin elasticity, and skin. Roughness, dryness of the skin, hair loss and reduced hair shine. The accumulation of these effects of sunlight is sometimes called photoaging.

Various cosmetic compositions have been studied to solve the problem of skin aging, and the wrinkle improvement effect of the skin has shown visible results in some. Retinoids, which are widely used to improve skin wrinkles, blemishes and pigmentation, have been reported variously, and clinical results of skin wrinkle removal for retinol have been reported. Retinol-containing cosmetics are used for skin wrinkles formed by sunlight. Effectively improved skin sagging and elasticity reduction. U.S. Patents 4,603,146 and 4,877,805 have examples of wrinkle improvement using retinol (vitamin A), which is effective in collagen synthesis and inhibition of collagen degradation by collagenase activity inhibition.

In addition, cosmetic compositions containing L-ascorbic acid (vitamin C), which are essential for the collagen synthesis process, have been reported to be effective for skin wrinkle improvement, sun protection, pigmentation such as blemishes, freckles, and black mushrooms (USP4,938,969 / USP4, 772,591 / EP0533667).

In addition, aha (α-Hydroxy acid: AHA) containing cosmetic composition that is effective in exfoliation has also been widely used to improve the skin by promoting the activity of the cells with the exfoliation.

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 are capable of producing 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 which 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. Therefore, the research for applying to the treatment, such as regeneration of damaged tissues, by using the differentiation capacity of stem cells are in progress.

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 beginnings 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. 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. In the present invention, it is intended to apply the stem cell culture extract to the cosmetic composition using these properties.

Stem cell cultivation method for producing human adipocyte conditioned media extracts is cultured for several generations of fat-depleted cells from adipose tissue to separate stem cells and then stem from stem cell culture. The protein produced by the cell can be obtained. This protein mixture contains EGF (epidermal growth factor), vEGF (vascular endothelial growth factor), TGF (transformation growth factor), HGF (hepatocyte growth factor), FGF (fibroblast growth factor), and IGF (insulin-like growth factor). It is reported that it contains about 150 growth factor protein components. 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. These growth factors are responsible for 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 applied to fibroblasts of the skin, the amount of collagen, a connective tissue produced by fibroblasts, increased more than five times, and the growth 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 cells that are multipotent and have the ability to differentiate into cells such as bone, cartilage, ligaments, fat, myocardium, and muscle in vitro as well as in vivo. 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. In view of its application as a cell for cell treatment, mesenchymal stem cells have a number of advantages over embryonic stem cells in that cell harvesting is relatively easy, safe, and self-transplantable. Because of the advantages mentioned above, the treatment of various diseases such as myocardial infarction, fracture, cartilage damage, and stroke using stem cells is being studied. However, unlike embryonic stem cells, stem cells are stem cells that can be utilized in culture in vitro, and have a very short lifespan and have a differentiation capacity for a short period of time. The limited lifespan of these stem cells is a major limitation in the utilization of the cells.

The use of stem cells in the cosmetic field is the use of growth factors as 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, the whitening effect on the subjects showing pigmentation phenomenon shows good results, ensuring strong efficacy and safety as a cosmetic raw material.

PDRN (Polydeoxyribonucleotide; PDRN) is a nucleotide fragment of a specific specification, which is sodium DNA extracted from salmon, a regression fish. PDRN, which is already used as an injectable drug and has proven its stability and efficacy, has tissue regeneration efficacy and stimulates A2 receptor, which is a skin regeneration signal, to promote the secretion of various growth factors, Vascular endothelial growth factor (VEGF) ) To produce capillaries, improve blood circulation, anti-inflammatory action and prevent capillary leakage. In addition, PDRN is always present in human cells and physiologically stimulates the regeneration and metabolic activity of fibroblasts to help maintain the best condition. PDRN (Polydeoxyribonucleotide) has received much attention as a result of research showing that it is effective in treating wounds such as burns by promoting skin regeneration without any special side effects, and it is possible to shorten wound healing time and activate cell regeneration by rapid tissue regeneration. In addition to promoting the production of non-collagenic proteins at the same time to form a normal matrix, to promote the differentiation of various cells (stem cells, fibroblast, osteoblast, chondrocyte, etc.) to improve VEGF induction and micro circulation, and also has an anti-inflammatory action. In addition, various researches on the treatment of hair loss have been progressed and emerging as a new treatment method using PDRN such as hair follicle cell regeneration, scalp capillary generation, and scalp tissue regeneration using PDRN. PDRN improves the blood circulation of the scalp and can observe newborn hair 4 to 6 weeks after the procedure. The patient's satisfaction is high. Also, the use of PDRN in hair transplantation compensates for the disadvantages of conventional PRP treatment. And edema that occurs after transplantation. In addition, PDRN regenerates and grows fibroblasts, which are skin stem cells, creates elastic fibers such as collagen and elastin, regenerates damaged skin quickly, and improves blood circulation to keep skin more elastic. It was judged to be a very effective way to maximize the effects of stem cells when used with various skin stem cells.

The present inventors found that the cosmetic composition comprising the culture extract of the human adipose stem cells and PDRN (Polydeoxyribonucleotide; PDRN) as an active ingredient has an excellent wrinkle improvement, whitening effect, skin elasticity enhancing effect, moisturizing effect and hair growth promoting effect. The present invention has been completed.

Korean Patent No. 10-1627562 discloses a cosmetic composition containing human adipose stem cell culture extract and hyaluronic acid. In addition, Korean Patent Publication No. 10-2017-0068857 discloses a composition for anti-inflammatory and skin regeneration using polydeoxyribonucleotide.

SUMMARY OF THE INVENTION An object of the present invention is to provide a cosmetic composition containing human adipose stem cell culture extract and PDRN, specifically, for improving wrinkles containing human adipose stem cell culture extract and PDRN. To provide a cosmetic composition for whitening, skin elasticity promotion, moisturizing and hair growth promoting.

The present invention is a cosmetic composition for improving wrinkles containing human fat stem cell culture extract and PDRN, the cosmetic composition is 0.01 to 10% by weight of human fat stem cell culture extract and 0.01 to 10% by weight of the total weight of the cosmetic It comprises 2.0 wt% PDRN, and the weight ratio of human fat stem cell culture extract and PDRN is 1: 1 to 10: 1.

The present invention is a whitening cosmetic composition containing human fat stem cell culture extract and PDRN, the cosmetic composition is 0.01 to 10% by weight of human fat stem cell culture extract and 0.01 to 2.0 to the total weight of the cosmetic It comprises weight% PDRN, and the weight ratio of the human fat stem cell culture extract and PDRN is 1: 1 to 10: 1.

The present invention is a cosmetic composition for enhancing skin elasticity containing human fat stem cell culture extract and PDRN, the cosmetic composition is 0.01 to 10% by weight of human fat stem cell culture extract and 0.01 to the total weight of the cosmetic To 2.0 wt% PDRN, and the weight ratio of the human adipose stem cell culture extract and PDRN (PDRN) is 1: 1 to 10: 1.

The present invention is a moisturizing cosmetic composition containing human fat stem cell culture extract and PDRN, the cosmetic composition is 0.01 to 10% by weight of human fat stem cell culture extract and 0.01 to 2.0 to the total weight of the cosmetic It comprises weight% PDRN, and the weight ratio of the human fat stem cell culture extract and PDRN is 1: 1 to 10: 1.

In addition, the present invention is a hair growth promoting cosmetic composition containing human fat stem cell culture extract and PDRN, the cosmetic composition is a human fat stem cell culture extract of 0.01 to 10% by weight relative to the total weight of the cosmetic and 0.01 to 2.0 wt% of PDRN, and the weight ratio of the human fat stem cell culture extract and PDRN of PDRN is 1: 1 to 10: 1.

By using a cosmetic composition containing human fat stem cell culture extract and PDRN according to the present invention, skin wrinkle improvement effect is superior to existing cosmetic composition containing only human fat stem cell culture extract or PDRN (PDRN) , Whitening effect, skin elasticity enhancing effect, moisturizing effect and hair growth promoting effect can be obtained.

The human fat stem cell culture extract includes a culture solution obtained by culturing fat stem cells and all extracts obtained by crushing the cultured fat stem cells.

The human adipose stem cell means a stem cell obtained from an adult except for embryonic stem cells, and includes all stem cells derived from the base of blood and tissue including bone marrow, re-blood, and peripheral blood, and the present invention is not limited thereto. no.

The active ingredients of the human adipose stem cell culture extract are mainly peptides and proteins that regulate cell growth, for example PDGF (platelet-derived growth factor), Basic FGF (basic fibroblast growth factor), KGF (keratin cell growth factor). ), TGF-β1 (transformation growth factor), HGF (hepatocyte growth factor), VEGF (vascular endothelial growth factor), collagen, fibronectin, SOD (antioxidant enzyme) and the like. Since the human stem cell culture extract contains the growth factors described above, skin cell constituent cells promote fibroblast migration, activation effect, skin regeneration and anti-aging effect, collagen synthesis increase, tyrosinase activity inhibition effect, melanin synthesis inhibition, hair Growth and contraction of hair follicle restoration can be expected.

Human fat stem cell culture extract of the present invention can be obtained by the method specified in US Pat. No. 6,153,432 (2000.11.28). First, adipose stem cells are separated from adipose tissue obtained through liposuction. This was suspended in substrate medium and inoculated in a culture vessel, followed by incubation for 24 hours at 37 ° C. in a CO ₂ incubator. After incubation for 3 days, adipocyte medium (adipocyte media) is added and differentiated into adipocytes. The differentiation of adipose stem cells begins at the beginning of the differentiation of small lipid droplets in the cytoplasm, gradually increasing the number of small lipid droplets, and gradually adding smaller lipid droplets to form large lipid droplets. Once fully matured, one lipid droplet occupies the entire cytoplasm and gradually increases in size during the maturation process. In the step of obtaining such adipose derived cells, the culture medium is serum-free DMEM medium, basic fibroblast growth factor (bFGF) and / or epidermal growth factor (EGF) 0.1 ~ The culture broth obtained by culturing in DMEM medium further comprising 100 ng / ml concentration was separated and purified and used as the adipose stem cell culture extract of the present invention.

PDRN of the present invention is an abbreviation of polydeoxyribonucleotide and is called PDRN because it shows pharmacological activity without transmitting genetic information unlike DNA fragments or general DNA. This is pure PDRN, an active form of 95% or higher purity obtained from salmon (Oncorhynchus keta) or trout (Oncorhynchus mykiss) semen and subjected to high heat treatment.The INCI name is Sodium DNA. Here, PDRN (polydeoxyribonucleotide) is composed of DNA polymer of specific size and when it is administered in biological tissues, it not only promotes DNA synthesis but also promotes rapid regeneration against excessive inflammatory reaction or tissue production. In particular, the rapid anti-inflammatory and tissue regeneration effect shortens the wound healing time, activates cell regeneration, contains collagen, activates the production of various proteins and promotes the differentiation of various cells.

It stimulates the skin regeneration signaling A2 receptor to promote the secretion of various growth factors, VEGF (Vascular endothelial growth factor vascular endothelial cell growth factor) to produce capillaries, improve blood circulation, anti-inflammatory action and prevent capillary leakage . In addition, PDRN is always present in human cells and physiologically stimulates the regeneration and metabolic activity of fibroblasts to help maintain the best condition. PDRN (Polydeoxyribonucleotide) has received much attention as a result of research showing that it is effective in treating wounds such as burns by promoting skin regeneration without any special side effects, and it has shortened wound healing time and activated cell regeneration by rapid tissue regeneration. In addition to promoting the production of non-collagenic proteins at the same time to form a normal matrix, to promote the differentiation of various cells (stem cells, fibroblast, osteoblast, chondrocyte, etc.) to improve VEGF induction and micro circulation, and also has an anti-inflammatory action. In addition, various researches on the treatment of hair loss have been progressed and emerging as a new treatment method using PDRN such as hair follicle cell regeneration, scalp capillary generation, and scalp tissue regeneration using PDRN. PDRN improves the blood circulation of the scalp and can observe newborn hair 4 to 6 weeks after the procedure. The patient's satisfaction is high. Also, the use of PDRN in hair transplantation compensates for the disadvantages of conventional PRP treatment. And edema that occurs after transplantation. In addition, PDRN regenerates and grows fibroblasts, which are skin stem cells, creates elastic fibers such as collagen and elastin, regenerates damaged skin quickly, and improves blood circulation to keep skin more elastic. It was judged to be a very effective way to maximize the effects of stem cells when used with various skin stem cells.

The cosmetic composition of the present invention may include 0.01 to 10.0% by weight of human fat stem cell culture extract and 0.01 to 2.0% by weight of PDRN based on the total weight of the cosmetic. In addition, the weight ratio of human adipose stem cell culture extract and PDRN may be 1: 1 to 10: 1, preferably 1: 1 to 5: 1.

According to a specific example of the present invention, the cosmetic composition of the present invention containing human fat stem cell culture extract and PDRN as an active ingredient, skin wrinkle improvement effect, whitening effect, skin elasticity enhancing effect, moisturizing effect and hair growth promotion It has a skin anti-aging effect such as effect.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples, but the scope of the present invention is not limited by these Examples and Experimental Examples.

For the cosmetic composition of the present invention, a cosmetic containing human fat stem cell culture extract and PDRN was prepared as in the following example.

Example 1 Preparation of Cosmetics Containing Human Adipose Stem Cell Culture Extract and PDRN

As shown in Table 1, the composition containing only human fat stem cell culture extract and PDRN (PDRN) and the composition containing only human fat stem cell culture extract (Comparative Example 1) and PDRN only (PDRN) A containing composition (Comparative Example 2) was prepared.

Unit: weight% ingredient Example 1 Comparative Example 1 Comparative Example 2 Human Adipose Stem Cell Culture Extract 10.0 12.0 - PDRN 2.0 - 12.0 A Arachidil Alcohol 3.0 3.0 3.0 Glyceryl Stearate 1.5 1.5 1.5 Squalane 5.0 5.0 5.0 Hydroxypolydesine 2.0 2.0 2.0 Trioctanoine 5.0 5.0 5.0 Polysorbate 80 1.0 1.0 1.0 Sorbitan stearate 0.5 0.5 0.5 Cyclopentasiloxane 3.0 3.0 3.0 Tocopheryl Acetate 0.2 0.2 0.2 BHT 0.05 0.05 0.05 B Purified water Remaining amount Remaining amount Remaining amount Concentrated glycerin 4.0 4.0 4.0 1,3-butylene glycol 2.0 2.0 2.0 Dimethicone / Vinyl Dimethicone Crosspolymer 0.4 0.4 0.4 EDTA-2Na 0.05 0.05 0.05 Incense, preservative Quantity Quantity Quantity system 100 100 100

Experimental Example 1. Improvement of skin wrinkles

1. Instrumental evaluation

In order to measure the skin wrinkle improvement effect (mechanical evaluation) on the cosmetic composition prepared by Example 1 according to the present invention, 30 women (age age 45.5 years) over 20 years old (A, B, C age group, respectively) Divided into, the cosmetic composition prepared according to the embodiment of Table 1 according to the present invention was applied to Example 1 in Group A, Comparative Example 1 in Group B, Comparative Example 2 in Group C.

A replica of the skin wrinkles was applied to each subject of group A, B, and C by applying a transparent silicone solution while applying it to the area of the upper arm 2 × 2 cm 2 for 6 weeks (2 times / day, 0.2 g / time). The skin wrinkles were measured using a skin wrinkle measuring instrument (SKIN VISIOMETER SV400, C + K Electronics GmbH. Germany). Three-dimensional analysis of the image of the replica with a CCD camera, wrinkles by the average wrinkle roughness (R _z ) of the following formula 1 which is the sum of the roughness (R _m : m is an integer of 1 or more) of each wrinkle divided by the number of wrinkles The improvement effect was analyzed. The test results are shown in Table 2 below.

Skin wrinkle improvement effect (6 weeks) Average wrinkle roughness (R _{z ,} ㎛) Example 1 Comparative Example 1 Comparative Example 2 T0 T6 ΔR _z T0 T6 ΔR _z T0 T6 ΔR _z Subject 1 125 51 -74 129 88 -41 128 84 -44 Subject 2 130 45 -85 126 50 -76 122 71 -51 Subject 3 90 32 -58 115 77 -38 110 90 -20 Subject 4 125 33 -92 119 58 -61 129 101 -28 Subject 5 128 20 -108 135 68 -67 126 53 -73 Subject 6 135 21 -114 126 42 -84 133 72 -61 Subject 7 145 35 -110 125 50 -75 125 89 -36 Subject 8 147 41 -106 127 87 -40 133 73 -60 Subject 9 124 26 -98 118 45 -73 126 46 -80 Subject 10 139 47 -92 129 61 -68 121 51 -70 Average -93.7 -62.3 -52.3

As can be seen in Table 2, after six weeks, the average wrinkle roughness (R _z ) of Example 1 according to the present invention was reduced by 93.7 ㎛ (p <0.01), Comparative Example 1 is 62.3 ㎛ (p <0.05 ), Comparative Example 2 is reduced by 52.3 μm (p <0.05), and the cosmetics containing human fat stem cell culture extract and PDRN according to the present invention contains only human fat stem cell culture solution Comparative Example 1 More about 33.5%, 44.2% than the comparative example 2 showed a wrinkle improvement effect.

Therefore, it can be seen that the cosmetic composition containing the human adipose stem cell culture extract and PDRN according to the present invention has a much more effective wrinkle improvement effect than the cosmetic containing only human adipose stem cell culture extract or PDRN alone. Can be.

2. Visual Evaluation

30 women over 20 years of age (mean evaluation) to measure the skin wrinkle improvement effect (visual evaluation) for the cosmetic composition prepared by the embodiment containing the human adipose stem cell culture extract and PDRN according to the present invention Age 45.5 years) are divided into three groups A, B, and C, respectively, and the cosmetic composition prepared according to the embodiment of Table 1 according to the present invention is Example 1 in Group A, Comparative Example 1 in Group B, Comparative Example 2 was applied to the C group, respectively.

After six weeks of application (2 times / day) around wrinkled eyes, each subject of group A, B, and C was classified into six grades for objective evaluation and subjective evaluation. The improvement degree (ΔW) of was measured. The results are shown in Tables 3 and 4 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 1 Comparative Example 1 Comparative Example 2 T0 T6 T0 T6 T0 T6 Subject 1 0 3 0 2 0 One Subject 2 0 3 0 2 0 One Subject 3 0 3 0 One 0 One Subject 4 0 2 0 2 0 2 Subject 5 0 3 0 One 0 One Subject 6 0 2 0 2 0 One Subject 7 0 3 0 2 0 2 Subject 8 0 2 0 2 0 One Subject 9 0 3 0 2 0 2 Subject 10 0 3 0 2 0 3 △ W (T6-T0) 2.7 1.8 1.5

Clinical effect of skin wrinkle improvement (subjective evaluation of subject) Skin wrinkle improvement (△ W) Example 1 Comparative Example 1 Comparative Example 2 T0 T6 T0 T6 T0 T6 Subject 1 0 3 0 2 0 2 Subject 2 0 2 0 2 0 2 Subject 3 0 3 0 One 0 One Subject 4 0 2 0 2 0 2 Subject 5 0 3 0 2 0 2 Subject 6 0 3 0 One 0 One Subject 7 0 3 0 3 0 2 Subject 8 0 3 0 2 0 2 Subject 9 0 2 0 2 0 One Subject 10 0 2 0 2 0 One △ W (T6-T0) 2.6 1.9 1.6

As can be seen from Table 3 and Table 4, in Example 1 containing the human adipose stem cell culture extract and PDRN according to the present invention, the objective evaluation of the skilled person and the subjective evaluation of the subject were 2.7 and 2.6, respectively. In the objective evaluation of the skilled person, an increase of 33.3% and 44.4% of Comparative Example 2 was higher than that of Comparative Example 1, and in the subjective evaluation of the subject, it was found to be 26.9% higher than Comparative Example 1 and 38.5% higher than Comparative Example 2. It can be seen that the cosmetics containing the adipose stem cell culture extract and PDRN have more effective wrinkle improvement than the cosmetics containing only human adipose stem cell culture extract or PDRN.

Experimental Example 2. Skin whitening effect (inhibitory effect on melanin production in melanocytes)

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. After 24 hours of incubation under 5% CO ₂ , the cells were isolated by treatment with 0.05% Trypsin containing 0.02% EDTA, and then incubated in a 50 ml T-flask for 48 hours. At this time, the cell number is 5.76 × 10 ⁶ cells / flask. Herein, the human adipose stem cell culture extract and PDRN of the present invention were diluted in DMEM medium at 1, 2, 5, 10, 20, 50, and 100 µg / ml, respectively, and treated with cultured melanoma cells 37 Incubate for 5 days at ℃. 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 5.

Inhibitory Effect of Melanin Synthesis in Melanosite density
(Μg / ml) Melanin production inhibition rate (%) Human Adipose Stem Cell Culture Extract + PDRN (5: 1) Human Adipose Stem Cell Culture Extract PDRN No treatment - - - One 10.0 4.8 5.5 2 18.2 12.8 11.8 5 35.5 23.7 28.1 10 43.9 26.2 33.9 20 51.6 29.9 39.3 50 62.9 38.1 51.0 100 82.1 45.3 65.5

As can be seen in Table 5, the human fat stem cell culture extract of Example 1 according to the present invention and PDRN mixed with a weight ratio of 5: 1 human fat stem cells of Comparative Example 1 The melanin synthesis inhibitory effect was higher than the treatment with only the culture extract and the treatment with PDRN of Comparative Example 2. Through this, when the human fat stem cell culture extract and PDRN of the present invention are used together, the melanin production of melanocytes is more effective than when the human fat stem cell culture extract or PDRN is used alone. It can be seen that the skin whitening effect is suppressed.

Experimental Example 3. Skin elasticity enhancing effect

Cosmetic Example 1 containing only the human adipose stem cell culture extract and PDRN of the present invention and cosmetics containing only the human adipose stem cell culture extract extract Example 1, comparing cosmetics containing only PDRN (PDRN) The skin elasticity improvement effect of Example 2 was measured.

Thirty females (average age 45.5 years) of 20 years or older (mean age 45.5 years) were divided into three groups A, B, and C, respectively, at a temperature of 24-26 ° C. and a humidity of 45%. The cosmetic composition was applied to group A, Comparative Example 1 to Group B, and Comparative Example 2 to Group C, respectively, for 6 weeks (2 times / day) around the eyes, and then, a skin elasticity measuring instrument (Cutometer SEM 575, C). + K Electronic Co., Germany) was used to measure skin elasticity. The test results are described in Table 6 below as the value of ΔR (before use) -R8 (after use) of the Cutometer SEM 575. R values indicate the nature of skin viscoelasticity.

Skin elasticity (after 6 weeks) Skin viscoelasticity improvement degree (△ R) Example 1 Comparative Example 1 Comparative Example 2 Subject 1 0.62 0.40 0.49 Subject 2 0.35 0.43 0.47 Subject 3 0.58 0.37 0.35 Subject 4 0.67 0.32 0.58 Subject 5 0.45 0.37 0.39 Subject 6 0.49 0.28 0.33 Subject 7 0.61 0.26 0.39 Subject 8 0.57 0.32 0.31 Subject 9 0.41 0.29 0.30 Subject 10 0.49 0.39 0.47 △ R 0.52 0.34 0.41

As can be seen from the results of Table 6, cosmetic Example 1 containing the human adipose stem cell culture extract and PDRN (PDRN) compared to Comparative Example 1 containing only the human adipose stem cell culture extract, skin elasticity 34.6%, PD Compared with Comparative Example 2 containing only R & D (PDRN) increased by 21.2% compared to the cosmetic containing only human fat stem cell culture extract and PDRN (PDRN) very effectively enhance skin elasticity.

Experimental Example 4. Moisturizing Effect

Cosmetic Example 1 containing only human fat stem cell culture extract and PDRN of the present invention and cosmetics containing only human fat stem cell culture extract Example 1 and cosmetics containing PDRN only The skin moisturizing effect for Example 2 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 according to Example 1 according to the present invention was divided into 30 groups (average age 45.5 years) of women over 20 years old (Average age 45.5 years), respectively. The cosmetic composition prepared by Comparative Example 2 in the silver group B was applied to the group C for 6 weeks (2 times / day) around the eyes, and then percutaneously using TEWAMETER TM210 (C + K electronic GmbH. Germany). The amount of water loss, ie TEWL (Transepidermal Water Loss), is measured as the TEWL value change ΔTEWL over time, and the change in electrical conductivity according to the epidermal moisture content is determined using CORNEOMETER CM 820 PC (C + K electronic GmbH. Germany). Moisturizing effect was measured by quantifying the moisture content of the skin from 0 to 150. The test results are shown in Table 7 below.

Skin moisturizing effect Test Product △ TEWL Corneometer value Example 1 6.8 139 Comparative Example 1 3.4 95 Comparative Example 2 4.9 118

As a result of the test, the ΔTEWL value of Example 1 was 6.8 g / hm ^2, and 6 weeks later, the transdermal moisture loss was increased by 50.0% compared to Comparative Example 1 and 27.9% compared to Comparative Example 2, which is very effective. .

In addition, it can be seen that even in the moisture measurement value (Corneometer value) measuring the moisture content of the skin, Example 1 is increased by 31.7% and 15.1% compared to Comparative Example 1, the skin is very moist.

Example 2 Preparation of Cosmetics Containing Human Adipose Stem Cell Culture Extract and PDRN

As shown in Table 8, the composition containing only human fat stem cell culture extract and PDRN (PDRN) and the composition containing only human fat stem cell culture extract (Comparative Example 3) and PDRN (PDRN) only A composition containing (Comparative Example 4) was prepared.

Unit: weight% ingredient Example 2 Comparative Example 3 Comparative Example 4 Human Fat Stem Cell Culture Extract 10.0 12.0 0.0 PDRN 2.0 0.0 12.0 A Cetostearyl alcohol 2.0 2.0 2.0 Glyceryl Stearate 1.5 1.5 1.5 Microcrystallin 0.7 0.7 0.7 Squalane 5.0 5.0 5.0 Liquid paraffin 3.0 3.0 3.0 Trioctanoine 5.0 5.0 5.0 Polysorbate 60 1.2 1.2 1.2 Sorbitan stearate 0.5 0.5 0.5 Cyclomethicone 3.0 3.0 3.0 Tocopheryl Acetate 0.2 0.2 0.2 BHT 0.05 0.05 0.05 Purified water Remaining amount Remaining amount Remaining amount B glycerin 4.0 4.0 4.0 1,3-butylene glycol 2.0 2.0 2.0 Xanthan Gum 0.1 0.1 0.1 EDTA-2Na 0.05 0.05 0.05 Incense, preservative Quantity Quantity Quantity system 100 100 100

Experimental Example  5. Hair growth promoting effect-Proliferation effect of dermal papilla cell

Rat vibrissa immortalized dermal papilla cells (Filsell W, et al., 1994) were treated with 100 units / mL penicillin-100 ug / mL streptomycin (Gibco Inc, NY, USA) and 10heat-inactivated fetal bovin serum (FBS; Gibco Inc, NY). , USA) was used to culture in DMEM (Hyclone Inc, USA) medium at 37 ℃, 5% CO ₂ incubator, subcultured once every three days. Proliferation of rat vibrissa immortalized dermal papilla cells was measured using MTT assay. Dermal papilla cells (1.0 × 10 ⁴ cells / mL) were placed in a 96well plate and incubated for 24 hours, then exchanged with serum-free DMEM medium for 24 hours, followed by Example 2, Comparative Example 3 and Comparative Example. Example 4 was treated at concentrations of 1, 10, 50 and 100 μg / ml respectively. The minoxidil sulfate (Sigma, USA), a positive control, was treated at a concentration of 1 μM. After incubation for 4 days, 50 uL of MTT (Sigma, MO, USA) is added and reacted for 4 hours. The supernatant is removed, dissolved in 200 μL of DMSO, and the absorbance is measured at 540 nm using a microplate reader (Amersh-am Pharmacia Biotech, NY, USA). The average absorbance values for each sample group were obtained, and the degree of proliferation was investigated by comparing with the absorbance values of the control group and shown in Table 9 below.

Proliferative effect of dermal papilla cells density
(μM) Proliferation of dermal papilla cells (%) minoxidil sulfate Example 2 Comparative Example 3 Comparative Example 4 No treatment - - - - One 36.0 40.1 23.9 30.1 10 79.9 62.5 41.4 48.8 50 92.5 77.7 54.2 65.4 100 97.2 88.9 60.8 72.4

As can be seen in Table 9, Example 2 was evaluated for the proliferative effect of dermal papilla dermal cells of Example 2 (composition containing human adipose stem cell culture extract and PDRN) of the dermal papilla It was found that the proliferative effect of the dermal cells is excellent. In particular, the proliferative effect of Dermal papilla cells of Example 2 showed a proliferative effect of dermal papillary dermal cells similar to minoxidil sulfate, a positive control. Therefore, it was found that the human fat stem cell culture extract and PDRN used in Example 2 exhibited an excellent hair growth promoting effect on hair follicle growth.

Experimental Example 6. Hair growth promoting effect-5α-reductase inhibitory effect

5α-reductase activity was assessed by converting [3H] testosterone (T) into [3H] dihydrotestosterone (DHT) using rat prostate 5α-reductase. Male 7 to 8 week old Wistar rats were anesthetized with ethyl ether, followed by cervical slaughter to remove the prostate gland, immediately stored in liquid nitrogen, and the required amount of prostate gland was taken out for each experiment. The prostate was placed in a glass homogeniger containing 4 ° C homogeniger buffer (20 mM potassium phosphate, pH6.5, 0.32 M sucrose, 0.1 mM dithiothreitol (DTT)) and ground until it became cloudy. After centrifugation (1000rpm, 10min, 4 ℃), cell free extract was obtained, followed by centrifugation (24,000rpm, 90min, 4 ℃) to remove the supernatant, and homogenizer buffe was added to the pellet and used as crude enzyme. It was. The 5α-reductase assay was performed as follows. Crude enzyme, sample and reaction buffer (40mM potassium phosphate, pH 6.5, 50uM NADPH, 120nCi [1,2,6,7-3H] T) were added to the microtube, and the final reaction solution was adjusted to 500 uL. The reaction was carried out for 30 minutes at. Add 1 mL of ethyl acetate, centrifuge (1000 g, 10 min), take the supernatant, transfer to a new microtube, and dry thoroughly using speed vac. 50 uL of ethyl acetate is added to the microtube, spotted on a TLC plate and developed twice under a developing solvent (50% cyclohexane, 50% ethyl acetate). Check T (testosterone) at 254nm with UV spectrometer, check DHT (dihydro testosterone) by spraying 10% sulfuric acid, cut out T and DHT site with scissors, put into each scintilation vial, add 10 mL cocktail and measure with β-counter do. The conversion rate of testosterone by 5α-reductase activity is calculated from the ratio of DHT / (T + DHT) and is shown in Table 10 below.

5α-reductase Inhibitory Effect density
(μM)  5a-reductase inhibitory effect (%) minoxidil sulfate Example 2 Comparative Example 3 Comparative Example 4 No treatment - - - - One 32.0 30.1 17.1 28.0 10 57.9 53.1 36.6 41.8 50 73.3 68.9 46.8 62.3 100 93.8 89.5 60.9 76.5

As can be seen in Table 10, Example 2 according to the present invention can be seen to exhibit a very good 5α-reductase inhibitory effect. In particular, 5α-reductase inhibitory effect when the human fat stem cell culture extract and PDRN were used together showed 5α-reductase inhibitory effect similar to minoxidil sulfate, a positive control. It can be seen that there is a promoting effect.

Experimental Example 7. Stability Test of Formulation

The stability test of the formulation for the cosmetic containing the human adipose stem cell culture extract of the present invention and PDRN (PDRN) was performed. Example 1, Example 2, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4 of the Table 1 in order to test the stability of the cosmetic constant temperature bath is kept constant at 45 ℃ After storing each in a transparent glass jar for 4 weeks, the degree of separation, discoloration and odor (specific odor) was measured and compared. The results are shown in Table 11 below. At this time, the product separation, discoloration, and degree of odor were evaluated by classifying into the following six grades.

Product discoloration evaluation criteria:

0: no change 1: very little separation (color change, odor)

2: Slightly separated (discolored, decolored) 3: Slightly separated (discolored, decolored)

4: severely separated (discolored, decolored) 5: extremely severely separated (discolored, decolored)

Temperature Separation discoloration Odor (special odor) Example 1 compare
Example 1 compare
Example 2 Example 1 compare
Example 1 compare
Example 2 Example 1 compare
Example 1 compare
Example 2 45 ℃ 0 0 0 0 0 0 0 0 0 4 ℃ 0 0 0 0 0 0 0 0 0 Temperature Separation discoloration Odor (special odor) Example 2 compare
Example 3 compare
Example 4 Example 2 compare
Example 3 compare
Example 4 Example 2 compare
Example 3 compare
Example 4 45 ℃ 0 0 0 0 0 0 0 0 0 4 ℃ 0 0 0 0 0 0 0 0 0

As can be seen in Table 11, Comparative Example 1, containing only the human adipose stem cell culture extract and PDRN of Example 1, Example 2 and human adipose stem cell culture extract of the present invention, Comparative Example 3 and Comparative Example 2 containing only PDRN and PDRN 4, it can be seen that there is no separation, discoloration and odor (specific odor) phenomenon is a stable base.

Experimental Example 8. Safety Test of the Formulation

Skin safety test for the cosmetic containing the human adipose stem cell culture extract and PDRN of the present invention was carried out.

Thirty subjects (mean age 40.3 years, age distribution 18 years-55 years) were formulated according to the Examples and Comparative Examples of the present invention according to Table 1 and Table 8 skin patch test on the upper arm using Haye's Test Chamber (Patch Test) ) Was performed. 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, 15 μg of Example 1, Example 2, Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4 were added dropwise to the chamber. It is fixed on the upper arm, which is the test site. The patch is applied for 24 hours, and after the patch is removed, the test site is marked with a marking pen and the test site is observed after 24 hours and 48 hours, respectively. Determination was performed 24 hours and 48 hours after patch removal, and skin reaction was determined according to the regulations of the International Contact Dermatitis Research Group (ICDRG) shown in Table 12 below. The test results are shown as skin stability in Table 13 below.

sign Criteria evaluation Mean score ±
+
++
+++
- Doubtful or slight reaction and erythema
Erythema + Induration
Erythema + Induration + Vesicle
Erythema + Induration + Bullae
Negative Smile stimulation
Light stimulation
Heavy stimulation
Strong stimulation
Non-irritating 0 to 0.9
1.0-2.9
3.0-4.9
5.0 or more
0

Elapsed time Example 1 Example 2 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 24 hours 0.0 0.0 0.0 0.0 0.0 0.0 48 hours 0.0 0.0 0.0 0.0 0.0 0.0

As shown in Table 13, as a result of skin safety test, in Example 1, Example 2, Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4, the average irritation degree is 0, and there is no irritation on the skin. It can be seen that it is a safe cosmetic.

Hereinafter, based on the results of the above experimental examples, to present a cosmetic composition containing human fat stem cell culture extract and PDRN (PDRN). However, the composition of the present invention is not intended to be limited to the following formulation examples.

Formulation example  1. Flexible Cosmetic (Skin Lotion)

Ingredient Parts by weight Human Adipose Stem Cell Culture Extract 3.0 PDRN 0.1 1,3-butylene glycol 6.0 glycerin 4.0 Oleyl alcohol 0.1 Polysorbate 20 0.5 ethanol 15.0 Benzophenol-9 0.05 Incense, preservative a very small amount Purified water to 100

Formulation example  2. Nutritional Cosmetics ( Milk Croissant )

Ingredient Parts by weight  Human Adipose Stem Cells 5.0  PDRN 0.5  Propylene glycol 6.0  glycerin 4.0  Triethanolamine 1.2  Tocopheryl Acetate 3.0  Floating paraffin 5.0  Squalane 3.0  Macadamia Nut Oil 2.0  Polysorbate 60 1.5  Sorbitan sesquioleate 1.0  Carboxyvinyl polymer 1.0  Bietchiti 0.01  IDT-2N 0.01  Incense, preservative a very small amount  Purified water to 100

Formulation example  3. Nutrition Cream

Ingredient Parts by weight  Human Adipose Stem Cell Culture Extract 10.0  PDRN 2.0  Cetostearyl alcohol 2.0  Glyceryl Stearate 1.5  Trioctanoine 5.0  Polysorbate 60 1.2  Sorbitan stearate 0.5  Squalane 5.0  Liquid paraffin 3.0  Cyclomethicone 3.0  Bietchiti 0.05  Delta-tocopherol 0.2  Concentrated glycerin 4.0  1,3-butylene glycol 2.0  Xanthan Gum 0.1  IDT-2N 0.05  Incense, preservative a very small amount  Purified water to 100

Formulation example  4. Massage Cream

Ingredient Parts by weight  Human Adipose Stem Cell Culture Extract 3.0  PDRN 0.3  Propylene glycol 6.0  glycerin 4.0  Triethanolamine 0.5  Beeswax 2.0  Tocopheryl Acetate 0.1  Polysorbate 60 3.0  Sorbitan sesquioleate 2.5  Cetearyl Alcohol 2.0  Liquid paraffin 30.0  Carboxy Vinyl Polymer 0.5  Incense, preservative a very small amount  Purified water to 100

Formulation example  5. Pack

Ingredient Parts by weight  Human Adipose Stem Cell Culture Extract 5.0  PDRN 0.5  Propylene glycol 2.0  glycerin 4.0  Carboxyvinyl polymer 0.3  ethanol 7.0  Fiji-40 Hydrogenated Castor Oil 0.8  Triethanolamine 0.3  Bietchiti 0.01  IDT-2N 0.01  Incense, preservative a very small amount  Purified water to 100

Claims (5)

delete A cosmetic composition for whitening containing human fat stem cell culture extract and sodium DNA, PDRN extracted from semen of salmon,
The cosmetic composition comprises 0.01 to 10% by weight of human fat stem cell culture extract and 0.01 to 2.0% by weight PDRN based on the total weight of the cosmetic composition,
The weight ratio of the human fat stem cell culture extract and PDRN (PDRN) is 5: 1, the cosmetic composition for whitening. delete delete As a cosmetic composition for promoting hair growth containing human fat stem cell culture extract and sodium DNA, PDRN, extracted from semen of salmon,
The cosmetic composition comprises 0.01 to 10% by weight of human fat stem cell culture extract and 0.01 to 2.0% by weight PDRN based on the total weight of the cosmetic composition,
The weight ratio of the human fat stem cell culture extract and PDRN (PDRN) is 5: 1, cosmetic composition for promoting hair growth.