KR20220164846A - 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 alleviating striae distensae comprising a culture medium of cord blood stem cells, ceramide, and milk peptides. The composition is excellent in preventing and alleviating striae distensae by a synergetic effect by the complex action of the culture medium of cord blood stem cells with skin regeneration, anti-inflammatory and collagen production effects, and ceramide and milk peptides with skin moisturizing effects. Therefore, the composition of the present invention can be used as a pharmaceutical composition for preventing or alleviating striae distensae and a cosmetic composition for preventing or alleviating striae distensae.

Description

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

The present invention relates to a composition for preventing or improving skin stretch marks comprising an 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 are thinned and rearranged parallel to the skin. Early lesions appear as red lines or bands on the skin, but if you look closely, they are slightly sunken than normal skin, so they feel bumpy when you touch them. These lesions are called striae rubra. The purple striae gradually turn white over time and become less distinct, giving way to wrinkled and atrophied skin, which is called striae alba.

As a specific method for preventing and mitigating stretch marks, once stretch marks are formed, it is more effective to treat them when they are in the early stages of purple lines rather than at the stage of completely scarred white lines. Devices used for the purpose of elasticity and moisturizing include a method using low-mid-high frequency or ultrasonic waves, and a vacuum suction device and an ultrasonic device using physical force. If you use a pulsed dye laser that effectively acts on red lesions, you can see the effect of treating stretch marks in the early stages, and other high frequency or pigment laser treatments are also used in parallel. On the other hand, in the white striatum stage where atrophy is common, various methods such as fractional scar laser, peeling, and MTS (fine needle acupuncture) treatment are used in parallel, but it is known that the effect is less than in the initial red stage.

In addition, there is a care for stretch marks using cosmetics. Although various treatments have been attempted to treat stretch marks, it is important to use effective cosmetics for care from the beginning because they are not perfect. Also, once they appear, early management is the most effective way to reduce the extent and extent of stretch marks. In the United States, Germany, and Italy, due to limitations in the aforementioned medical methods, cream formulations that can be used in general are being researched, developed, produced, and sold, and some are used for cosmetics containing aroma oil, moisturizing, and elasticity ingredients. is being studied. Applying these specialized cosmetics consistently reduces skin irritation and replenishes moisture, which can reduce skin stress even with sudden hormonal and body shape changes, helping to care for stretch marks.

To date, methods for treating stretch marks include application of tretinoin ointment, laser treatment such as pulsed dye laser (PDL) and fractional laser, and cosmetics that help relieve stretch marks are available on the market. However, most of the cosmetics that help relieve stretch marks use cell growth factors and some vegetable extracts, and are only used for other auxiliary external applications in dermatological procedures. When used alone, the cosmetic composition has insufficient effect on preventing and improving stretch marks. there is

Umbilical cord blood refers to the blood remaining in the placenta and umbilical cord after delivery of the fetus. Umbilical cord blood contains a lot of undifferentiated hematopoietic stem cells with relatively high proliferative capacity, so hematological reconstruction like bone marrow transplantation is possible with umbilical cord blood stem cells obtained from one person's cord blood. Umbilical cord blood, as a source of stem cells, is commercially more advantageous than embryonic stem cells, which are practically impossible to use, and is easier to commercially use in that it does not require artificially harvesting bone marrow or fat from the adult body like adult stem cells. can be said to be

Cell activating substances secreted from umbilical cord blood stem cells are largely composed of several proteins that have a mutual relationship with collagen as a center, and it can be confirmed that they induce the synthesis of collagen or strengthen the binding between collagen fibers.

Accordingly, the inventors of the present invention developed a composition excellent in preventing and improving skin stretch marks using a culture solution containing a substance secreted from cord blood stem cells.

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

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

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

In one embodiment, the ceramide may be included in an amount greater than 0 and 1% by weight based on the total weight of the composition.

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

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

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

In one embodiment, the pharmaceutical and cosmetic composition can inhibit MMP-1 gene expression in dermal 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 peptides promotes cell growth and proliferation of skin cells, and decreases the expression of MMP-1 to increase collagen synthesis. It has been found that it is suitable for preventing and improving stretch marks. In addition, it was confirmed that when a stretch mark cream was prepared and used with the composition of the present invention including the above three components, skin regeneration, moisturizing, and elasticity improvement effects were exhibited.

Therefore, the mixed composition of cord blood stem cell culture medium (HSCM), ceramide, and milk peptides 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 above effects, and should be understood to include all effects that can be inferred from the description of the present invention or the configuration of the invention described in the claims.

Figure 1 shows that in skin keratinocytes (HaCaT) (a) treatment with HSCM by concentration (1, 3, 5, 10, 30% by weight), (b) milk peptide and ceramide combination by concentration (described The concentration is the result of evaluating the cell proliferation effect by treatment with milk peptide and ceramide at each treatment concentration of 0.001, 0.005, 0.01, 0.05, and 0.1% by weight, respectively).
Figure 2 is (a) a microscopic image and (b) a graph showing the result of evaluating the cell proliferation efficacy of the combination, HSCM, and a mixture thereof (HSCM + combination) in skin keratinocytes (HaCaT).
3 is a graph showing (a) a microscope image and (b) a numerical result of evaluating the wound healing efficacy of the combination, HSCM, and a mixture thereof (HSCM + combination) in skin keratinocytes (HaCaT).
Figure 4 is an experimental result of evaluating the collagen biosynthesis promoting effect of the combination, HSCM, and a mixture thereof (HSCM + combination) in skin keratinocytes (HaCaT).
Figure 5 is after inducing the expression of MMP-1 induced by TNF-α in dermal keratinocytes (HaCaT) HSCM, the combination, and the mixture (HSCM + combination) MMP-1 gene expression inhibitory effect evaluated by RT-PCR is a result
6 is a result of evaluating the effect of improving skin stretch marks of a cosmetic composition containing HSCM, milk peptide, and ceramide on a test subject (pregnant woman).

The present invention provides a pharmaceutical composition and a cosmetic composition for preventing or improving skin stretch marks containing cord blood stem cell culture medium, ceramides, and milk peptides.

In the present invention, the cord blood stem cells are stem cells isolated from umbilical cord blood from the umbilical cord, and may contain hematopoietic stem cells that produce white blood cells, red blood cells, platelets, etc., and cartilage, bone, muscle and nerve It may also contain mesenchymal stem cells that make up the back. The cord blood stem cells are not limited to a specific cell line. In addition, cord blood stem cells can be easily constructed by those skilled in the art.

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

The compositions may include the cord blood stem cell culture medium in an amount greater than 0 and 30% by weight, preferably 0.01 to 20% by weight, more preferably 0.1 to 10% by weight, and more preferably 1% by weight, based on the total weight. can include

The compositions may contain ceramides and milk peptides in an amount greater than 0 and 1% by weight, respectively, preferably 0.001 to 0.1% by weight, more preferably 0.01% by weight, respectively, based on the total weight.

The above compositions can promote the proliferation of skin keratinocytes. It is known that the cord blood stem cell culture medium contains various basic protein components, among which epidermal growth factor (EGF) is included. EGF is a factor that promotes the growth of epithelial cells and is known to promote the growth of epithelial cells by inducing cell division by transmitting signals through EGF receptors in cell membranes. Cord blood stem cell culture medium containing growth promoting proteins other than EGF itself can induce cell proliferation of keratinocytes. can appear

The compositions can increase the secretion of collagen. It is known that when a lot of collagen is secreted from keratinocytes, the elasticity of the skin improves. Cord blood stem cell culture medium alone can increase collagen secretion in skin keratinocytes, but when ceramide and milk peptides are added, collagen secretion in skin keratinocytes can be significantly increased by synergy.

In the above compositions, when MMP-1 gene expression, which is one of the genes that promote the expression of enzymes that degrade collagen in skin keratinocytes due to inflammation, is increased, the anti-inflammatory substances contained in the umbilical cord blood stem cell culture medium reduce inflammation, and MMP -1 The expression of the gene can be suppressed. Accordingly, the expression of collagen may be increased.

Hereinafter, the present invention will be described in more detail through examples and test examples. However, the following examples and test examples are intended to illustrate 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)

Frozen cord blood was thawed, diluted with alpha-minimum essential medium (αMEM) medium, centrifuged to harvest monocytes, cultured to obtain cord blood-derived pluripotent stem cells, washed three times with PBS, and placed in an activated medium. was diluted and activated for 24 hours. After the activation of the stem cells, the cells were washed three times with PBS, a culture medium was added, and the culture medium was collected by replacing the medium once every two days. The collected culture medium was filtered through a filter (Top Filter system, Corning), and then stored in a refrigerator or freezer before use. A detailed HSCM manufacturing method is described in Patent Registration No. 10-2082745.

2. Experiment details

In order to impart a moisturizing effect to the prepared cream formulation containing the three components, a composition containing the three components was prepared by combining HSCM (umbilical cord blood stem cell culture medium), milk peptides, and ceramide. At this time, the cream formulation was prepared as a commonly used cream formulation, and hereinafter, only the contents corresponding to HSCM, ceramide and milk peptides in the total composition were described.

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

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

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

After inoculating keratinocytes (HaCaT) in a 12-well plate with ⁴ 1X10 cells per well, the cells were stabilized for 24 hours. The next day, the cells were washed three times with PBS, replaced with FBS-free medium, treated with HSCM in each well, and treated with milk peptides and ceramides simultaneously and cultured for up to 3 days.

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

As shown in FIG. 1, as the concentration of HSCM treatment increased from 1 to 30% by weight, the number of keratinocytes increased, whereas when treated with a combination of milk peptide and ceramide, the concentration of milk peptide and ceramide was 0.001, respectively. As the amount increased to ~ 0.05% by weight, the amount of keratinocytes increased and then decreased at 0.1% by weight.

(2) Evaluation of synergistic effect of skin cell proliferation of mixtures containing HSCM and milk peptide + ceramide

In this experiment, the synergistic effect of the mixture was confirmed by evaluating the proliferation promoting efficacy of HSCM, milk peptide + ceramide, and mixtures thereof in keratinocytes.

After inoculating keratinocytes (HaCaT) in a 12-well plate with ⁴ 1X10 cells per well, the cells were stabilized for 24 hours. The next day, the cells were washed three times with PBS, replaced with FBS-free medium, and 1 wt% of HSCM, 0.01 wt% of milk peptide and ceramide were added to each well, and a mixture thereof (HSCM 1 wt%, milk 0.01% each of peptide and ceramide) were treated and cultured for up to 3 days.

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

As shown in Figure 2, HSCM and milk peptide + ceramide treatment group did not show a noticeable difference in the proliferation of skin keratinocytes (HaCaT), whereas in the mixture treatment group, the degree of cell proliferation was three times higher than that of the HSCM alone treatment group It was found that an increase in

(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 a mixture thereof have a wound healing ability in skin keratinocytes.

Skin keratinocytes (HaCaT) were inoculated in 1X10 ⁶ per well in a 6-well plate. When confluency reached 100%, scratches were made on the surface of the plate using a tip, and washed three times with PBS. Replace with FBS-free medium, and in each well, 1 wt% of HSCM, 0.01 wt% of milk peptide and ceramide, and a mixture thereof (1 wt% of HSCM, 0.01 wt% of each of milk peptide and ceramide) ) were treated and cultured for 24 hours. Then, after removing the medium and washing with PBS three times, the state of cell migration was confirmed using a microscope.

All three or more images of the scratch part were taken, and the same part was taken in the same way after 24 hours. The photographed results are shown in Fig. 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 FIG. 3(b). All experiments were repeated three times.

As shown in FIG. 3, the wound healing ability was slightly improved in the HSCM and milk peptide + ceramide alone treatment group, but it was confirmed that the cell healing ability was further increased in the mixture treatment group with a synergistic effect.

(4) Collagen type I biosynthesis promoting effect test (ELISA method)

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

In order to obtain protein from keratinocytes, keratinocytes (HaCaT) were inoculated with 1X10 ⁵ per well and then stabilized for 24 hours. Thereafter, the medium was removed, washed three times with PBS, and each well was treated with HSCM, milk peptide + ceramide, and a mixture thereof 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.

In order to quantify the total protein and collagen obtained, each protein was quantified with a BCA protein assay kit (Pierce) to equalize the total amount of each protein. After reacting proteins prepared in the same amount using a procollagen type I C-peptide EIA kit (Takara), the amount of collagen type I among total proteins was confirmed with an ELISA reader (BioTek). The results are shown in FIG. 4 .

As shown in Figure 4, it was confirmed that the synthesis of collagen type I was significantly increased in the mixture treatment group. From this result, it was assumed that the cell proliferation rate tended to increase with the increase in collagen synthesis.

(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 degrade collagen, such as MMP-1. In this experiment, the inhibitory activity of HSCM, milk peptide + ceramide, and mixtures thereof on TNF-α-induced MMP-1 expression was confirmed by RT-PCR analysis.

The process of RNA isolation and acquisition from keratinocytes was performed as follows. Skin keratinocytes (HaCaT) were inoculated with 1X10 ⁵ per well and then stabilized for 24 hours. After starvation of the cells in FBS-free medium for 6 hours, each well was treated with HSCM, milk peptide + ceramide, and a mixture thereof together with TNF-α (50 ng/ml) for 24 hours.

Then, the medium was removed, washed three times with PBS, and total RNA was extracted from the cultured cells using RNAzol B reagent for gene expression analysis. After dissolving the cells by adding 1 ml of RNAzol B reagent to denature the tissue, transfer each to a 1.5 ml tube, add 200 μl of chloroform, and vortex for 20 seconds to ensure complete mixing. After reacting at room temperature for 15 minutes, the supernatant was obtained by centrifugation at 14,000 rpm for 20 minutes, and the same amount of isopropyl alcohol was added, inverted, 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% RNA ethanol at 14,000 rpm for 10 minutes, and dried for 5 minutes using an aspirator. For the dried RNA sample, 25 μl of distilled water treated with 0.1% DEPC (diethyl pyrocarbonate) was added and reacted at 55 ° C for 10 minutes to dissolve the pellet and used as a sample for cDNA synthesis. 5 μl of it was diluted 20 times O.D. using a spectrophotometer. Concentration and purity of RNA were measured at 260/280 nm.

For cDNA synthesis, single-stranded cDNA synthesis was performed by mixing 1 μl of oligo-d(T) primer (100 pmol) with 1 μg of extracted total RNA, followed by reaction at 65° C. for 10 minutes, followed by rapid cooling. To this template, 2 μl each of 10 mM dNTP (TaKaRa Bio Inc., Japan), 0.1 M DTT and 5x RT buffer (10 mM Tris-Cl, 50 mM KCl, 2.5 mM MgCl₂) was added and M-MLV RTase (BioNeer, Korea) after adding 100 units, the total amount was calibrated to be 20 μl using distilled water treated with DEPC. The sample was terminated by inactivating reverse transcriptase through synthesis reaction at 25 ℃ for 5 minutes and 42 ℃ for 1 hour, followed by reaction at 72 ℃ for 15 minutes.

RT-PCR reaction was performed using the cDNA synthesized through the above process and the primers of each gene. Reaction conditions were 1 μl of template, 0.5 μl of each primer (10 pmol), 0.5 μl of 2.5 mM dNTP, 10X PCR buffer [10 mM Tris-Cl (pH 8.3), 50 mM KCl, 2.5 mM MgCl ₂ ] 2.5 μl, Taq polymerase After adding 2 units/μl, the PCR reaction was performed by correcting the total amount to 25 μl using sterile distilled water. 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). Relative quantification of band detection intensity was corrected based on the detection intensity of GAPDH (glyceraldehyde-3-phosphate dehydrogenase), a house keeping gene, and is shown in FIG. 5 .

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

(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 the effect of improving skin with stretch marks was confirmed.

For test subjects (pregnant women), the prepared cream was applied to stretch marks twice a day for 4 weeks. Images were taken before and after the experiment started, which is shown in FIG. 6 . Whether or not the stretch mark area was reduced was measured using an Optical Modulation Analyzer from Tektronix before and after applying the cream, and the area of 3.000 mm ² of the stretch mark measurement area was measured and shown in Table 1.

As shown in FIG. 6, it was confirmed that the stretch mark area was visually alleviated, and it was confirmed that the stretch mark area was significantly reduced compared to before cream application even from the numerical values in Table 1.

P1 P2 P3 P4 P5 before application 3 mm ² 3 mm ² 3 mm ² 3 mm ² 3 mm ² after application 2.352 mm ² 2.412 mm ² 2.284 mm ² 2.312 mm ² 2.43 mm ²

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

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims (14)

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

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