KR102618453B1 - Composition for preventing or improving striae distensae comprising culture medium of cord blood stem cell - Google Patents

Abstract

The present invention relates to a composition for preventing or improving skin stretch marks containing umbilical cord blood stem cell culture medium, ceramide, and milk peptide. Cord blood stem cell culture medium with skin regenerative, anti-inflammatory, and collagen production effects and a skin moisturizing effect. Since the synergy effect of the combined action of ceramide and milkpetide is excellent in preventing and improving skin stretch marks, the composition of the present invention can be used as a pharmaceutical composition for preventing or improving skin stretch marks and a cosmetic composition for preventing or improving skin stretch marks.

Description

Composition for preventing or improving skin stretch marks comprising culture medium of cord blood stem cells {Composition for preventing or improving striae distensae comprising culture medium of cord blood stem cell}

The present invention relates to a composition for preventing or improving skin stretch marks containing umbilical cord blood stem cell culture medium.

Stretch marks are atrophic linear band lesions that appear on the skin in areas damaged by pulling force. Histologically, the epidermis is atrophied, and the collagen fibers in the dermis become thinner and rearrange parallel to the skin. Early lesions appear as red lines or bands on the skin, but if you look closely, they are slightly sunken compared to normal skin and feel bumpy when touched. These lesions are called striae rubra. As time goes by, the purple striae gradually turn white and become less distinct, turning into wrinkled and shriveled skin. This is called white striae (striae alba).

As a specific way to prevent and alleviate stretch marks, once stretch marks appear, it is more effective to treat them when they are in the early stages of purple striae rather than at the stage of completely scarred white striae. Care using procedures is generally used to treat the skin in cosmetics. Devices used for elasticity and moisturizing purposes include methods using low, medium and high frequencies or ultrasonic waves, and vacuum aspirators and ultrasonic devices that use physical force. Using a pulsed dye laser that works effectively on red lesions can be effective in treating early stretch marks, and can also be combined with high-frequency treatment or pigment laser treatment. On the other hand, in the white striae stage, where atrophy occurs frequently, various methods such as fractional scar laser, dermabrasion, and MTS (micro-needle) treatment are treated in parallel, but it is known to be less effective than in the early red stage.

Additionally, there is stretch mark care using cosmetics. Although various treatments are being attempted to treat stretch marks, they are not perfect, so it is important to use effective cosmetics to care for them from the beginning. Also, once they appear, early management is the most effective way to reduce the extent and extent of stretch marks. In countries such as the United States, Germany, and Italy, due to the limitations of the aforementioned medical methods, cream preparations that can be generally used are being researched, developed, produced, and sold. In some cases, they are used in cosmetics containing aromatic oils and moisturizing and elastic ingredients. is being studied. By consistently applying these professional cosmetics, you can reduce skin irritation and replenish moisture, which helps in treating stretch marks by reducing skin stress despite sudden hormonal and body shape changes.

To date, methods to treat stretch marks include applying tretinoin ointment and laser treatments such as pulsed dye laser (PDL) and fractional laser, and cosmetics that help relieve stretch marks are available on the market. However, most cosmetics that help relieve stretch marks use cell growth factors and some plant extracts and are only used for external use as an auxiliary to dermatological procedures. When used alone, the cosmetic composition has insufficient effect to prevent and improve stretch marks. There is.

Cord blood refers to the blood remaining in the placenta and umbilical cord after delivery of the fetus. Umbilical cord blood contains many undifferentiated hematopoietic stem cells with a relatively high proliferative capacity, and hematological reconstruction, like bone marrow transplantation, is possible with cord blood stem cells obtained from a single person's cord blood. Umbilical cord blood is commercially more advantageous than embryonic stem cells, which cannot realistically be used as a source of stem cells, and is easier to use commercially in that it does not require artificially collecting bone marrow, fat, etc. from the adult body like adult stem cells. It can be said that it is.

The cell-activating substances secreted from umbilical cord blood stem cells are largely composed of several proteins that have a mutual relationship centered on collagen, and can be confirmed to induce the synthesis of collagen or strengthen the bonds between collagen fibers.

Accordingly, the present inventors developed a composition that is effective in preventing and improving skin stretch marks using a culture medium containing substances secreted from umbilical cord blood stem cells.

The problem to be solved by the present invention is to provide a composition that is excellent in preventing and improving stretch marks.

In order to achieve the above problem, in one embodiment of the present invention, a pharmaceutical composition and a cosmetic composition for preventing or improving stretch marks containing umbilical cord blood stem cell culture medium, ceramide, and milk peptide are provided.

In one embodiment, the cord blood stem cell culture medium may be included in an amount ranging from 0 to 30% by weight based on the total weight of the composition.

In one embodiment, the ceramide may be included in an amount ranging from 0 to 1% by weight based on the total weight of the composition.

In one embodiment, the milk peptide may be included in an amount ranging from 0 to 1% by weight based on the total weight of the composition.

In one embodiment, the pharmaceutical and cosmetic composition can promote the proliferation of skin keratinocytes.

In one embodiment, the pharmaceutical and cosmetic composition can promote collagen secretion from skin keratinocytes.

In one embodiment, the pharmaceutical and cosmetic composition can inhibit MMP-1 gene expression in skin keratinocytes.

According to the present invention, it was confirmed that the mixed composition of umbilical cord blood stem cell culture medium (HSCM), ceramide, and milk peptide promotes cell growth and proliferation in skin cells, and increases collagen synthesis by reducing the expression of MMP-1. It has been found to be suitable for preventing and improving stretch marks. In addition, it was confirmed that the use of a stretch mark cream using the composition of the present invention containing the above three ingredients exhibits skin regeneration, moisturizing, and elasticity improvement effects.

Therefore, the mixed composition of cord blood stem cell culture medium (HSCM), ceramide, and milk peptide of the present invention can be usefully used as an external agent for preventing and improving stretch marks in the pharmaceutical and cosmetic fields.

The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the description or claims of the present invention.

Figure 1 shows skin keratinocytes (HaCaT) treated with (a) HSCM at different concentrations (1, 3, 5, 10, 30 wt%), and (b) a combination of milk peptide and ceramide at different concentrations (as described). The concentration is the treatment concentration of milk peptide and ceramide (0.001, 0.005, 0.01, 0.05, and 0.1% by weight, respectively) and is the result of evaluating the cell proliferation effect.
Figure 2 shows (a) a microscope image evaluating the cell proliferation efficacy of the combination, HSCM, and a mixture thereof (HSCM + combination) in skin keratinocytes (HaCaT) and (b) the results quantified and presented graphically.
Figure 3 shows (a) a microscope image evaluating the wound healing efficacy of the combination, HSCM, and a mixture thereof (HSCM + combination) in skin keratinocytes (HaCaT) and (b) the results quantified and presented graphically.
Figure 4 shows the results of an experiment evaluating the effect of promoting collagen biosynthesis of the combination, HSCM, and its mixture (HSCM + combination) in skin keratinocytes (HaCaT).
Figure 5 shows the inhibition effect of HSCM, combination, and mixture thereof (HSCM + combination) on MMP-1 gene expression by RT-PCR after inducing the expression of MMP-1 induced by TNF-α in skin keratinocytes (HaCaT). This is one result.
Figure 6 shows the results of evaluating the skin stretch mark improvement effect of a cosmetic composition containing HSCM, milk peptide, and ceramide on test subjects (pregnant women).

The present invention provides a pharmaceutical composition and a cosmetic composition for preventing or improving skin stretch marks, including umbilical cord blood stem cell culture medium, ceramide, and milk peptide.

In the present invention, the cord blood stem cells are stem cells isolated from umbilical cord blood, and may contain hematopoietic stem cells that produce white blood cells, red blood cells, and platelets, and cartilage, bones, muscles, and nerves. It may also contain mesenchymal stem cells that produce the back. The cord blood stem cells are not limited to specific cell lines. Additionally, umbilical cord blood stem cells can be easily constructed by those skilled in the art.

The umbilical cord blood stem cells may secrete growth factors and anti-inflammatory factors into the culture medium during culturing. These growth factors and anti-inflammatory factors can increase the growth and function of skin cells and produce various effects such as anti-inflammatory effects, collagen production, wrinkle improvement, wound healing, and whitening. Therefore, the umbilical cord blood stem cell culture medium can regulate the inflammatory response of the skin, promote the proliferation of skin cells, and enhance collagen production, and the ceramide and the milk peptide have a skin moisturizing effect, so they act in combination to prevent skin stretch marks. It can be effective in prevention and improvement.

The compositions may contain umbilical cord blood stem cell culture medium in an amount of more than 30% by weight based on the total weight, preferably 0.01 to 20% by weight, more preferably 0.1 to 10% by weight, and more preferably 1% by weight. It can be included.

The compositions may contain ceramide and milk peptide in an amount of 0 to 1% by weight, preferably 0.001 to 0.1% by weight, and more preferably 0.01% by weight, based on the total weight.

The compositions can promote the proliferation of skin keratinocytes. It is known that umbilical cord blood stem cell culture medium contains various basic protein components, among which is epidermal growth factor (EGF). EGF is a factor that promotes the growth of epithelial cells and is known to induce cell division and promote the growth of epithelial cells by transmitting signals through the EGF receptor on the cell membrane. Cord blood stem cell culture media containing growth promoting proteins other than EGF itself can induce cell proliferation of skin keratinocytes, but when ceramide and milk peptide are added, the synergistic effect on cell proliferation of skin keratinocytes is achieved. It may appear.

The compositions can increase the secretion of collagen. It is known that when more collagen is secreted from skin keratinocytes, skin elasticity improves. Cord blood stem cell culture medium itself can increase collagen secretion from skin keratinocytes, but when ceramide and milk peptide are added, collagen secretion from skin keratinocytes can increase significantly due to synergy.

The compositions increase the expression of the MMP-1 gene, one of the genes that promote the expression of enzymes that decompose collagen in skin keratinocytes due to inflammation, and the anti-inflammatory substances contained in the umbilical cord blood stem cell culture medium reduce inflammation and MMP -1 Can suppress gene expression. Accordingly, the expression of collagen may increase.

Hereinafter, the present invention will be described in more detail through examples and test examples. However, the following examples and test examples are for illustrating the present invention, and the scope of the present invention is not limited thereto.

<Example>

1. Preparation of cord blood stem cell culture medium (HSCM)

The frozen cord blood was thawed, diluted with αMEM (alpha-minimum essential medium), centrifuged to harvest monocytes, cultured to obtain cord blood-derived pluripotent stem cells, washed three times with PBS, and cells cultured in activation medium. was diluted and activated for 24 hours. After stem cell activation was completed, the cells were washed three times with PBS, culture medium was added, and the culture medium was collected by replacing the medium every two days. The collected cultures were each filtered using a filter (Top Filter system, Corning) and then stored in refrigerated and frozen storage. The detailed HSCM manufacturing method is described in Patent Registration No. 10-2082745.

2. Experiment details

In order to provide a moisturizing effect to the three-component cream formulation, HSCM (cord blood stem cell culture medium), milk peptide, and ceramide were mixed to prepare a composition containing three components. At this time, the cream formulation was prepared as a commonly used cream formulation, and below, only the contents corresponding to HSCM, ceramide, and milk peptide in the total composition are described.

(1) Evaluation of the cell proliferation effect of HSCM and milk peptide + ceramide in skin keratinocytes (HaCaT)

Human keratinocyte cell line (HaCaT) were cultured in a 5% CO ₂ incubator at 37°C using Dulbecco's Modified Eagle's Medium containing 10% FBS (fetal bovine serum) and 1% antibiotic, and incubated for 3 days. Subculture was performed once.

In this experiment, the degree of proliferation of skin keratinocytes (HaCaT) was evaluated to confirm the proliferation-promoting effect of HSCM and milk peptide + ceramide in skin keratinocytes.

Skin keratinocytes (HaCaT) were inoculated into a 12-well plate at 1×10 ⁴ cells per well, and the cells were stabilized for 24 hours. The next day, the cells were washed three times with PBS, then replaced with FBS free medium, treated with HSCM in each well, and treated with milk peptide and ceramide simultaneously and cultured for up to 3 days.

After 3 days of culture, the difference in cell growth was confirmed through images, and the number of cells was counted and quantified using a hemocytometer, which is shown in Figure 1. All experiments were repeated three times.

As shown in Figure 1, the number of skin keratinocytes increased as the HSCM treatment concentration increased from 1 to 30% by weight, while when treated with a combination of milk peptide and ceramide, the concentrations of milk peptide and ceramide were 0.001, respectively. As the amount increased to ~0.05% by weight, skin keratinocytes increased and then tended to decrease at 0.1% by weight.

(2) Evaluation of the synergistic effect on skin cell proliferation of a mixture containing HSCM and milk peptide + ceramide

In this experiment, the proliferation-promoting effect of HSCM, milk peptide + ceramide, and their mixture in skin keratinocytes was evaluated to confirm the synergistic effect of the mixture.

Skin keratinocytes (HaCaT) were inoculated into a 12-well plate at 1×10 ⁴ cells per well, and the cells were stabilized for 24 hours. The next day, the cells were washed three times with PBS, then replaced with FBS free medium, and each well was injected with 1% by weight of HSCM, 0.01% by weight of milk peptide and ceramide, and a mixture thereof (1% by weight of HSCM, 0.01% by weight of milk). Each was treated with peptide and ceramide (containing 0.01% each) and cultured for up to 3 days.

After 3 days of culture, the difference in cell growth was confirmed through images, and the number of cells was counted and quantified using a hemocytometer, and is shown in Figure 2. All experiments were repeated three times.

As shown in Figure 2, there was no noticeable difference in the proliferation of skin keratinocytes (HaCaT) in the HSCM and milk peptide + ceramide treatment group, whereas in the mixture treatment group, the degree of cell proliferation was 3 times that of the HSCM treatment group alone. It was confirmed that there had been an increase.

(3) Evaluation of healing efficacy for wounds such as stretch marks

In this experiment, an experiment was conducted to evaluate whether HSCM, milk peptide + ceramide, and their mixture had wound healing ability in skin keratinocytes.

Skin keratinocytes (HaCaT) were inoculated into a ⁶ -well plate at 1X106 cells per well. When confluency reached 100%, a scratch was made on the plate surface using a tip and washed three times with PBS. Replace with FBS free medium and add 1% by weight of HSCM, 0.01% by weight of milk peptide and ceramide in each well, and a mixture thereof (1% by weight of HSCM and 0.01% by weight of milk peptide and ceramide each). ) were each treated and cultured for 24 hours. After removing the medium, the cells were washed three times with PBS, and the mobility of the cells was checked using a microscope.

Images of the scratch area were taken of three or more parts, and 24 hours later, the same part was taken using the same method. The photographed results are shown in Figure 3(a). To quantify the degree of healing, each image was calculated using ImageJ software (http://imagej.nih.gov/ij/index.html), and the results are shown in Figure 3(b). All experiments were repeated three times.

As shown in Figure 3, the wound healing ability was found to be slightly improved in the HSCM and milk peptide + ceramide treatment group alone, but the cell healing ability was confirmed to be further increased through a synergistic effect in the mixture treatment group.

(4) Test for the effect of promoting collagen type I biosynthesis (ELISA method)

It is known that when more collagen is secreted from skin keratinocytes (HaCaT), skin elasticity improves. In this experiment, the amount of collagen type I synthesized by HSCM, milk peptide + ceramide, and their mixture was confirmed by ELISA analysis.

To obtain proteins from keratinocytes, skin keratinocytes (HaCaT) were inoculated at 1×10 ⁵ per well and stabilized for 24 hours. Afterwards, the medium was removed and washed three times with PBS, and then each well was treated with HSCM, milk peptide + ceramide, and their mixture for 48 hours. After 48 hours, the medium was removed, washed three times with PBS, and the cells were collected, dissolved in lysis buffer, and refrigerated centrifugation (4°C, 13,000 rpm, 30 min) to remove unnecessary cell shells.

To quantify the total protein and collagen obtained, each protein was quantified using a BCA protein assay kit (Pierce) to equalize the total protein amount. Equal amounts of proteins were reacted using the procollagen type I C-peptide EIA kit (Takara), and then the amount of collagen type I among the total proteins was confirmed using an ELISA reader (BioTek). The results are shown in Figure 4.

As shown in Figure 4, it was confirmed that the synthesis of collagen type I was significantly increased in the mixture treatment group. Based on these results, it was assumed that the proliferation rate of cells tended to increase as collagen synthesis increased.

(5) Evaluation of the degree of inhibition of MMP-1 gene expression (RT-PCR method)

Tumor Necrosis Factor-alpha (TNF-α) secreted by skin keratinocytes (HaCaT) is known to act on skin cells to promote the expression of enzymes that decompose collagen, such as MMP-1. In this experiment, the inhibitory activity of HSCM, milk peptide + ceramide, and their mixture on TNF-α-induced MMP-1 expression was confirmed through RT-PCR analysis.

The RNA isolation and acquisition process from keratinocytes was performed as follows. Skin keratinocytes (HaCaT) were inoculated at ^1X105 per well and stabilized for 24 hours. After starvating the cells in FBS free medium for 6 hours, each well was treated with HSCM, milk peptide + ceramide, and their mixture together with TNF-α (50 ng/ml) for 24 hours.

Next, the medium was removed, washed three times with PBS, and total RNA within the cells was extracted from the cultured cells using RNAzol B reagent for gene expression analysis. After adding 1 ml of RNAzol B reagent to dissolve the cells and denature the tissue, each was transferred to a 1.5 ml tube, and 200 μl of chloroform was added and vortexed for 20 seconds to mix thoroughly. After reacting at room temperature for 15 minutes, the supernatant was obtained by centrifugation at 14,000 rpm for 20 minutes, inverted by adding an equal amount of isopropyl alcohol, and left at room temperature for 10 minutes. The sample was centrifuged at 14,000 rpm for 15 minutes to obtain an RNA pellet, washed with 70% ethanol for RNA by centrifugation at 14,000 rpm for 10 minutes, and dried using an aspirator for 5 minutes. The dried RNA sample was used as a sample for cDNA synthesis after adding 25 μl of distilled water treated with 0.1% DEPC (diethyl pyrocarbonate) and reacting at 55°C for 10 minutes to dissolve the pellet. 5 μl of the sample was diluted 20 times. O.D. using a spectrophotometer. The concentration and purity of RNA were measured at 260/280 nm.

For cDNA synthesis, 1 μg of extracted total RNA was mixed with 1 μl of oligo-d(T) primer (100 pmol), reacted at 65°C for 10 minutes, and then rapidly cooled. To this template, 2 μl each of 10mM dNTP (TaKaRa Bio Inc., Japan), 0.1M DTT and 5x RT buffer (10mM Tris-Cl, 50mM KCl, 2.5mM MgCl₂) were added and M-MLV RTase (BioNeer, After adding 100 units (Korea), the total amount was adjusted to 20 μl using distilled water treated with DEPC. The sample was synthesized at 25°C for 5 minutes and 42°C for 1 hour, followed by reaction at 72°C for 15 minutes to inactivate reverse transcriptase.

RT-PCR reaction was performed using cDNA synthesized through the above process and primers for each gene. Reaction conditions were 1 μl of template, 0.5 μl (10 pmol) of each _primer , 0.5 μl of 2.5 mM dNTP, 2.5 μl of 10 After adding 2 units/μl, the total amount was adjusted to 25 μl using sterilized distilled water and a PCR reaction was performed. The amplification products obtained from the PCR reaction were subjected to electrophoresis using a 1% agarose gel, and the detection intensity of each PCR product was analyzed using an image analysis system (Kodak EDAS290). The relative quantitative value of the band detection intensity is corrected based on the detection intensity of GAPDH (glyceraldehyde-3-phosphate dehydrogenase), a house keeping gene, and is shown in Figure 5.

As a result of the experiment, it can be seen that the expression of MMP-1 was significantly reduced in the group treated with HSCM alone, and the expression of MMP-1 was also decreased in the group treated with milk peptide + ceramide. In addition, it was confirmed that MMP-1 expression in the mixture treatment group was decreased compared to the HSCM treatment group alone.

(6) Evaluation of stretch mark improvement effect in pregnant women

In this experiment, a skin regeneration cream-type cosmetic composition containing HSCM, milk peptide, and ceramide was prepared and its effectiveness in improving stretch mark skin was confirmed.

The cream prepared for the test subjects (pregnant women) was applied to the stretch marks area twice a day for 4 weeks. Images were taken before and after the experiment began, which are shown in Figure 6. The reduction in the area of stretch marks was shown in Table 1 by measuring the area of 3.000 mm ² of the stretch mark area before and after applying the cream using an optical modulation analyzer from Tektronix.

As shown in Figure 6, it was confirmed with the naked eye that the stretch mark area was alleviated, and the numbers in Table 1 also confirmed that the stretch mark area was significantly reduced compared to before applying the cream.

P1 P2 P3 P4 P5 Before application 3 mm ² 3 mm ² 3 mm ² 3 mm ² 3 mm ² After application ^{2.352mm2 2.412mm2} 2.284 ^{mm2 2.312mm2 2.43mm2}

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive. For example, each component described as unitary may be implemented in a distributed manner, and similarly, components described as distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the claims described below, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be construed as being included in the scope of the present invention.

Claims (14)

A pharmaceutical composition for preventing or improving skin stretch marks comprising umbilical cord blood stem cell culture medium, ceramide, and milk peptide. The pharmaceutical composition according to claim 1, wherein the cord blood stem cell culture medium is contained in an amount of more than 0 to 30% by weight based on the total weight of the composition. The pharmaceutical composition according to claim 1, wherein the ceramide is contained in an amount of 0 to 1% by weight based on the total weight of the composition. The pharmaceutical composition according to claim 1, wherein the milk peptide is contained in an amount ranging from 0 to 1% by weight based on the total weight of the composition. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition promotes the proliferation of skin keratinocytes. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition promotes collagen secretion from skin keratinocytes. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition inhibits MMP-1 gene expression in skin keratinocytes. A cosmetic composition for preventing or improving skin stretch marks comprising umbilical cord blood stem cell culture medium, ceramide, and milk peptide. The cosmetic composition according to claim 8, wherein the cord blood stem cell culture medium is included in an amount of more than 0 to 30% by weight. The cosmetic composition according to claim 8, wherein the ceramide is contained in an amount ranging from 0 to 1% by weight based on the total weight of the composition. The cosmetic composition according to claim 8, wherein the milk peptide is contained in an amount ranging from 0 to 1% by weight based on the total weight of the composition. The cosmetic composition according to claim 8, wherein the cosmetic composition promotes the proliferation of skin keratinocytes. The cosmetic composition according to claim 8, wherein the cosmetic composition promotes collagen secretion from skin keratinocytes. The cosmetic composition of claim 8, wherein the cosmetic composition inhibits MMP-1 gene expression in skin keratinocytes.