KR101742629B1 - Cosmetic composition for promoting the stemness of adipose-derived stem cell containing apple stem cell extracts - Google Patents

Abstract

The present invention provides a composition for promoting stem cell activity in adipose derived stem cells containing an apple stem cell extract as an active ingredient. The composition of the present invention is useful for increasing the adipose tissue for cosmetic or cosmetic purposes by enhancing the stem cell characteristics of adipose stem cells by containing an apple stem cell extract as an active ingredient. Thus, the composition of the present invention improves the skin volume and enables the low-molecular components capable of permeating the skin to effectively transmit through the skin, thereby promoting the regeneration of the fat-derived stem cells and restoring the function of the damaged subcutaneous fat. It can be utilized as a cosmetic or molding composition for enhancing the sensation and can be used as a composition for external application for skin for increasing skin volume.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a composition for promoting stem cell growth of an adipose-derived stem cell containing an apple stem cell extract. [0002]

The present invention relates to a stem cell growth promoting composition for maintaining and promoting the characteristics of adipose derived stem cells.

The skin is the largest organ of the human body, accounting for about 16% of our body volume, 1.8 m ^{2 in} area, 2 ~ 4 mm in thickness, weighing about 3 kg, and the epidermis, dermis, . Such skin is in direct contact with the external environment and plays an important protective layer for protecting the human body from various harmful factors such as harmful microorganisms and ultraviolet rays, and at the same time, it has a biochemical function necessary for metabolism of the whole body. It is an indispensable institution. In particular, the subcutaneous fat layer below the dermis is composed of adipocyte lobules, which acts as a cushion to absorb the protection of the internal organs of the skin against external heat, and to absorb the nutrients of the triglyceride (Lipid droplet), which is stored in a cell organelle and used as an energy source when necessary. However, as the age increases, the skin becomes damaged due to natural factors and various external stimuli. In particular, the activity and expression of collagen (collagen) and elastic fiber (elastin) At the same time, the fat production in the subcutaneous fat is reduced, so that the original function of each constituent cell in the skin is not achieved properly. Therefore, the most widely used method is fat graft surgery, which uses a syringe having a needle with a tulip needle-shaped tip and grabs its own fat, to be. However, it has been reported that 40 to 60% of the transplant volume is resorbed into the human body after the fat transplant surgery, and fat transplant surgery in areas where the skin is thin and the subcutaneous fat is small causes problems such as cell mass being touched And the survival rate and the survival period of transplanted fat are not as expected, and re-transplantation may be necessary.

There is a growing demand for a composition capable of sufficiently increasing the subcutaneous fat layer while replacing the fat transplant surgery with these problems. In addition, the basic structure of the skin is maintained by subcutaneous fat tissue, and since this subcutaneous fat tissue plays a role in the volume and intensity of the skin, it is more effective than the method of imparting elasticity to the dermis or epidermis of the skin outer- Increasing the volume of the tissue can be a good solution to maintain the volume of the skin.

Therefore, it has been necessary to develop a composition capable of increasing the volume of adipose tissue in order to maintain and enhance the volume of the skin.

In order to solve the above problems, it is an object of the present invention to provide a composition capable of enhancing stem cell characteristics of fat-derived stem cells capable of increasing adipose tissue and increasing skin volume.

In order to accomplish the above object, the present invention provides a composition for promoting stem cell activity of an adipose derived stem cell comprising an apple stem cell extract as an active ingredient.

The present invention also provides a cosmetic or molding composition for enhancing skin volume sensitivity comprising an apple stem cell extract as an active ingredient.

The present invention also provides a composition for external application for skin, which comprises an apple stem cell extract as an active ingredient.

The composition of the present invention is useful for increasing the adipose tissue for cosmetic or cosmetic purposes by enhancing the stem cell characteristics of adipose stem cells by containing an apple stem cell extract as an active ingredient. Thus, the composition of the present invention can be utilized as a beauty or molding composition for enhancing skin volume by promoting regeneration of fat-derived stem cells, restoring the function of damaged subcutaneous fat, It can be utilized as a composition for external application for skin.

FIG. 1 is a graph showing the cytotoxicity of the composition of the present invention. The human adipose stem cells (hADSCs) were treated with apple stem cell extract at a concentration of 0.0001 to 10% As shown in FIG.
FIG. 2 is a graph comparing the expression patterns of CD29, 44, 73, 90, and 105, which represent stem cell characteristics of adipose-derived stem cells, after oxidative stress was applied to adipose-derived stem cells and treated with apple stem cell extract to be.
FIG. 3 is a graph comparing the expression patterns of proteins expressing stem cell characteristics of adipose derived stem cells by treating the stem cell extract of apple after the oxidative stress was applied to the adipose-derived stem cells.
FIG. 4 is a graph showing growth factors secreted by adipose-derived stem cells by treating the stem cell extract of adulthood after oxidative stress is applied to adipose-derived stem cells.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for promoting stem cell activity in adipose derived stem cells containing an apple stem cell extract as an active ingredient. The term "stemness" refers to a state of stem cells having self-renewal ability and homeostasis of stem cells, and the term "stem cell growth" means that stem cell activity is maintained or the activity of stem cells is increased.

The method for obtaining the stem cell extract of the present invention is as follows.

When the outer surface of an apple is hurt by sharpness, it tries to regenerate because of the peculiar response of the plant. At this time, the regeneration site is transferred to a culture plate, and cells are proliferated through a general cell culture method. Then, the cells grow on the medium, and a large amount of apple stem cells is obtained. The reason for choosing apples among many plants is because the proliferation of stem cells of apple is faster than other plants and it is easy to use because it can obtain a large number of stem cells.

In one embodiment of the present invention, the adipose derived stem cell (ADSC) is human adipose derived stem cells (hADSCs).

In one embodiment of the present invention, the composition increases fat tissue and increases the volume feel on the skin. The composition promotes the differentiation of adipose-derived stem cells into adipocytes to produce lipid droplets. So it increases the fat tissue and increases the volume feeling on the skin of the specific area.

In one embodiment of the present invention, the composition comprises at least one of a cluster of differentiation 29 (hereinafter referred to as CD29) or a cluster of differentiation 44 (hereinafter referred to as CD44), a cluster of differentiation 73: hereinafter CD73), Cluster of Differentiation 90 (hereinafter referred to as CD90), or Cluster of Differentiation 105 (hereinafter referred to as CD105). The increase in the expression level of CD29, CD44, CD73, CD90 or CD105 as compared with the negative control means that the activity of adipose stem cells is increased. CD29 is another marker for integrin beta 1, a marker marker for adipose derived stem cells, and CD44 increases the activity of adipose-derived stem cells through Wnt signaling activity. CD73 is an enzyme that converts a nucleotide to a nucleoside in the name of a 5'-nucleotidase and is a marker marker for adipose derived stem cells. CD90 is another name for Thi1 Is a representative marker marker of stem cells, and CD105 is a marker of adipose-derived stem cells as part of the receptor of TGFb in other names of Endoglin.

In one embodiment of the present invention, the composition increases the expression amount of adipose-derived stem cell growth factor. The growth factors include, but are not limited to, neurotrophin 3 (NT3), neurotrophin 4 (NT4), platelet-derived growth factor receptor A (PDGF) Ra, a platelet-derived growth factor receptor B, a transforming growth factor b2 or a transforming growth factor b3, (Hereinafter referred to as " TGFb3 "). The increase in the expression level of the growth factor as compared with the negative control means that the number of adipose stem cells is increased. NT3 and NT4 are growth factors involved in cell division and growth. PDGF Ra, PDGF Rb, TGFb2 and TGFb3 are receptors for PDGF, a growth factor involved in cell division and growth.

In one embodiment of the present invention, the composition may contain 0.0001 to 10% by weight, more specifically 0.0001 to 0.1% by weight, more specifically 0.0001 to 0.001% by weight of apple stem cell extract, can do. If the content is less than 0.0001% by weight, the effect is insignificant. If the content is more than 10% by weight, there is a problem of exhibiting cytotoxicity.

An embodiment of the present invention provides a cosmetic or molding composition for enhancing skin volume sensation comprising an apple stem cell extract as an active ingredient.

In one embodiment of the present invention, the composition may contain 0.0001 to 10% by weight, more specifically 0.0001 to 0.1% by weight, more specifically 0.0001 to 0.001% by weight of apple stem cell extract, can do. If the content is less than 0.0001% by weight, the effect is insignificant. If the content is more than 10% by weight, there is a problem of exhibiting cytotoxicity.

The composition may be used, for example, for the purpose of removing wrinkles and wrinkles and maintaining elasticity by enhancing the volume feeling on the face such as breast enlargement through breast tissue formation, micro fat grafting, autologous fat transplantation.

The composition may be formulated into a unit dosage form suitable for administration to a patient in the body according to conventional methods in the pharmaceutical field. As a formulation suitable for such purpose, an injectable preparation or a preparation for topical administration is preferably used as the parenteral administration preparation. The injectable formulations are preferably isotonic aqueous solutions or suspensions. However, at this time, diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like can be used together.

The thus prepared preparation of the present invention can be administered parenterally according to a conventional method, for example, directly to a specific site. At this time, the composition is injected using a syringe. The daily dose of the composition is 1 × 10 ⁵ to 1 × 10 ⁷ cells / kg body weight, preferably 5 × 10 ⁵ to 5 × 10 ⁶ cells / kg body weight, and can be administered once or several times . It should be understood, however, that the actual dosage of the active ingredient should be determined in light of various relevant factors such as the amount of fat cells to differentiate and proliferate, the route of administration, the weight of the patient, age and sex, And are not intended to limit the scope of the invention in any way.

One embodiment of the present invention provides a composition for external application for skin, which comprises an apple stem cell extract as an active ingredient.

In one embodiment of the present invention, the composition may contain 0.0001 to 10% by weight, more specifically 0.0001 to 0.1% by weight, more specifically 0.0001 to 0.001% by weight of apple stem cell extract, can do. If the content is less than 0.0001% by weight, the effect is insignificant. If the content is more than 10% by weight, there is a problem of exhibiting cytotoxicity.

The composition for external application for skin may be a cosmetic composition or a pharmaceutical composition.

The formulation of the cosmetic composition is not particularly limited and may be appropriately selected according to the purpose. For example, it can be manufactured by a formulation such as a soft lotion, a nutritional lotion, a massage cream, an essence, a nutrition cream, a pack, and the like, but is not limited thereto.

The pharmaceutical compositions may be in a transdermal dosage form such as, but not limited to, lotions, ointments, gels, creams, patches or sprays.

In the composition for external application for skin according to each formulation, components other than the composition of the present invention may be mixed and selected without difficulty by those skilled in the art depending on the formulation or use purpose of other external preparation for skin, etc. In this case, A synergistic effect can occur.

Further, when the composition according to the present invention is used as a pharmaceutical composition, it may further contain a preservative, a stabilizer, a wetting agent or an emulsifying accelerator, a pharmaceutical adjuvant such as a salt or buffer for controlling the osmotic pressure, and other therapeutically useful substances And can be formulated into various oral or parenteral dosage forms according to conventional methods.

It should be understood that the actual dosage of the active ingredient should be determined in light of various relevant factors such as the severity of the symptoms, the route of administration selected, the age, sex, weight and health status of the subject.

Generally, the dosage of active ingredient is in the range of 0.001 mg / kg / day to about 2000 mg / kg / day. A more preferred dosage is 0.5 mg / kg / day to 2.5 mg / kg / day.

The application site of the composition for promoting stem cell function of the adipose-derived stem cells according to the present invention includes all the adipose tissues of the body and may include, for example, . In addition, the effect of increasing the amount of adipose tissue may be an effect of increasing the volume of skin due to the effect of increasing adipose tissue for cosmetic or cosmetic purposes, but is not limited thereto.

Hereinafter, the present invention will be described in more detail with reference to the following Examples and Test Examples, which should not be construed as limiting the scope of the present invention.

[Test Example 1] Cytotoxicity of apple stem cell extract in human adipose-derived stem cells

Fat-derived stem cells were subcultured in a 75 cm ² T-flask and cultured in a CO ₂ incubator (CO2 Incubator) at 37 ° C and 5% CO ₂ . Typically, passage of cells was performed in the second or third cycle.

Human adipose derived stem cells (hADSCs) were purchased from Lonza, Inc. (Walkersville, MD, USA) and cultured according to Lonza's guidelines. To determine the cytotoxicity of the composition, the concentration was 0.0001-10%, and the degree of cytotoxicity was confirmed using a CCK-8 kit (Dojindo). As shown in FIG. 1, about 10% cell division enhancement was observed without treatment of cytotoxicity at a concentration of 0.001%.

[Test Example 2] Measurement of effect of apple stem cell extract on expression level of adipose-derived stem cell marker marker gene

The oxidative stress of the adipose-derived stem cells was treated with hydrogen peroxide (H ₂ O ₂ ) at a concentration of 200 uM for 24 hours, and the composition was added to the medium in an amount of 0.001% in order to evaluate the stem cell growth effect of the adipose-derived stem cells . The cells were washed with 2 ml of phosphate buffered saline (PBS) and then treated with triazol reagent (Invitrogen, Carlsbad, Calif., USA) RNA was isolated from the cells.

(Agilent 2100 BioAnalyzer, Agilent Technologies, Santa Clara, Calif., USA) after purification of the separated RNA with an RNA kit (QIAGEN RNeasykt, QIAGEN, Valencia, CA) Were used to confirm the quality of the RNA. CDNA was synthesized from the RNA using a reverse transcription kit (Superscript Reverse Transcriptase (RT) kit, Invitrogen, Carlsbad, Calif.), And the cDNA was synthesized using real time reverse transcription polymerase chain reaction -RT-PCR).

The expression patterns of the CD29, 44, 73, 90 and 105 genes in adipose-derived stem cells were measured using a TaqMangene Expression Assay Kit (Applied Biosystems, Foster City, Calif.) (Applied Biosystems, Foster City, Calif .; Hs00559595_m1, CD44 Gene primer: Hs01075861_m1, CD73 gene primer: Hs01573922_m1, CD90 gene primer: Hs00174816_m1, CD105 gene primer: Hs00923996_m1). The results are shown in FIG. The expression level of each gene was increased by about 50% as compared with the negative control. These increases were statistically significant, and statistical tests were verified by both Student's t test, indicating a significant difference when the p-value was less than 0.05.

[Test Example 3] Measurement of fat-derived stem cells through flow cytometry

Human adipose tissue-derived stem cells were separated from 75 cm ² Ti flasks with 0.25% Trypsin-EDTA, and the cells were allowed to settle for 5 minutes at 1500 rpm. After removing the culture medium, 3 ml of blocking buffer: blocking buffer, 2% The cells were suspended by adding fetal calf serum (FCS) and 2% bovine serum albumin (BSA) KGM-2.

Cells (approximately 10 ⁶ cells) were applied for 15 min at 4 ° C and then stained with FITC (Fluorescein isothiocyanate) -containing CD44 antibody (Cat No. 560977) (BD Pharmingen, CA, US) and PE- (cat. No. 559882) (BD Pharmingen, CA, US) with 100 ng / ml-Cyanine was added to each well and reacted at 4 ° C for 45 minutes. FITC Rat IgG2a, k isotype (Cat No. 555742) and PE-Cy5 mouse IgG1 k isotype (Cat No. 555750) were also added to the cells used as the negative control, Gt; C < / RTI > for 45 minutes.

After the reaction was completed, the cells were washed twice with 3 ml of washing buffer (2% Bovine Serum Albumin (BSA) KGM-2), and then the cells were suspended. Thereafter, changes in the number of cells expressing CD29, 44 in adipose derived stem cells were observed with FACSCalibur (BD Bioscience, San Jose, Calif., U. S.) and are shown in FIG.

Negative control group Apple stem cell extract Derived stem cells expressing CD29 2558 3030 increase % 34% Derived stem cells expressing CD44 3112 6363 increase % 104%

Referring to FIG. 3 and Table 1, CD29 was 34% as compared to negative control. CD44 was increased by 104% .

[Test Example 4] Expression of Growth Factor in Fat-Derived Stem Cell by Apple Stem Cell Extract

Growth factor array (Raybio) was performed to understand the effect of the stem cell extract of the present invention on the expression of growth factors of adipose derived stem cells.

Growth factor arrays were performed by collecting the conditioned medium of adipose - derived stem cells cultured under each condition. 2 ml of blocking buffer was treated at room temperature for 30 minutes and then 1 ml of the culture was treated at room temperature for 2 hours. The cells were then washed 5 times with 2 ml of wash buffer and reacted with biotin-conjugated anti-growth factor antibody for 2 hours at room temperature. After washing with 2 ml of washing buffer 5 times for 5 minutes, 2 ml of HRP-conjugated streptoavidin was treated for 2 hours, washed with washing buffer, and reacted with detection buffer to obtain LAS-3000 (Fujifilm) Respectively.

As shown in FIG. 4, the growth factors decreased by 10% or more due to oxidative stress and increased by 10% or more by treatment with the stem cell extract of apple, NT3, NT4, PDGF Ra, PDGF Rb, TGFb2 and TGFb3 were analyzed. These increases were statistically significant, and statistical tests were verified by both Student's t test, indicating a significant difference when the p-value was less than 0.05.

Formulation Examples and Formulation Examples for the composition of the present invention are illustrated below.

[Formulation Example 1] Flexible longevity

The formulated softening water containing the above-mentioned Example 1 was prepared according to a conventional production method with the composition shown in Table 2 below.

ingredient Content (% by weight) Betaine 3.0 Natto sword 3.0 Cellulose sword 0.005 ethanol 10.0 Polyoxyethylene hardened castor oil 0.2 Tocopheryl acetate 2.0 Apple stem cell extract 10 antiseptic a very small amount Pigment a very small amount incense a very small amount Purified water Balance

[Formulation Example 2] Nutritional lotion

The nutritional lotion formulation containing the above Example 1 was prepared according to a conventional preparation method with the composition shown in Table 3 below.

ingredient Content (% by weight) Cetyl ethylene hexanoate 4.0 Cetostearyl alcohol 1.0 Chin type monostearyl stearate 1.0 Squalane 0.5 Apple stem cell extract 5.0 Polysorbate 60 1.5 Sorbitan sesquioleate 0.5 glycerin 5.0 Triethanolamine 0.5 Carboxyvinyl polymer 0.2 antiseptic a very small amount Pigment a very small amount incense a very small amount Purified water Balance

[Formulation Example 3] Cream

The cream formulations containing the above Example 1 were prepared according to conventional manufacturing methods with the compositions shown in Table 4 below.

ingredient Content (% by weight) Glyceryl stearate 3.0 Polysorbate 60 1.0 Cetostearate 2.5 Wax 1.0 Squalane 2.0 Sorbitan sesquioleate 0.5 Cetyl ethyl hexanoate 0.5 Liquid paraffin 5.0 Active ingredient mixture 5.0 glycerin 3.0 Propylene glycol 3.0 Apple stem cell extract 5.0 antiseptic a very small amount Pigment a very small amount incense a very small amount Purified water Balance

[Formulation Example 1] Tablets

100 mg of the stem cell extract of the present invention, 400 mg of lactose, 400 mg of corn starch and 2 mg of magnesium stearate were mixed, and tablets were prepared by tableting according to a conventional preparation method of tablets.

[Formulation Example 2]

100 mg of the stem cell extract of the present invention, 400 mg of lactose, 400 mg of corn starch and 2 mg of magnesium stearate were mixed and filled in gelatin capsules according to the conventional preparation method of capsules to prepare capsules.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (11)

delete delete delete delete delete delete delete A composition for cosmetic or molding for the promotion of subcutaneous fat, comprising an apple stem cell extract as an active ingredient. 9. The method of claim 8,
Wherein the composition contains 0.0001 to 10% by weight of an apple stem cell extract based on the total weight of the composition. A composition for external application for skin for promoting subcutaneous fat, which comprises an apple stem cell extract as an active ingredient. 11. The method of claim 10,
Wherein the composition contains 0.0001 to 10% by weight of an apple stem cell extract based on the total weight of the composition.

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