KR101778746B1 - Composition for promoting the differentiation of human mesenchymal stem cell - Google Patents

Abstract

The present invention relates to a composition for promoting the differentiation of human mesenchymal stem cells into adipocytes. More particularly, the present invention relates to a composition for promoting the differentiation of human mesenchymal stem cells into fat cells, Lipid droplets to improve the volume and elasticity of the skin and improve wrinkles.

Description

[Technical Field] The present invention relates to a composition for promoting the differentiation of human mesenchymal stem cells,

The present invention relates to a composition for promoting the differentiation of human mesenchymal stem cells into adipocytes. More particularly, the present invention relates to a composition for promoting the differentiation of human mesenchymal stem cells into fat cells, Lipid droplets to improve the volume and elasticity of the skin and improve wrinkles.

The subcutaneous fat layer below the dermis is composed of adipocyte lobules. It acts as a cushion to absorb the protection of the internal organs of the skin against external heat and absorbs the nutrients into the form of triglyceride (Lipid droplet), which is stored in the organelle and used as an energy source if 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 decreased, so that the original function of each constituent cell is not properly performed in the whole skin in the end, and skin aging phenomenon such as wrinkle formation due to loss of elasticity occurs.

In modern society, especially for women, skin is a major indicator of their health, and skin that loses elasticity as it gets older is becoming a sensitive issue for women. Particularly, the skin of the face area is thinner than other skin areas and is liable to lose its elasticity due to factors such as weight and age. In addition, the loss of elasticity and fat in the face caused by metabolic abnormalities such as hormones, blood, fat and bones, which are mainly caused by menopausal women, make women feel a great loss of femininity. Increasing the volume of local organizations is becoming a solution to these very sensitive issues for modern women, and many women are trying to maintain or increase the firmness of their skin through plastic surgery with side effects and economic burdens. do.

To improve the elasticity of the skin, a widely used method is fat transplant surgery. Fat transplant surgery is a surgical operation using a syringe with a tulip needle shaped tip and a needle that is not sharp. These fat grafts can be applied to the areas that have been lost due to the aging skin, wrinkles of the aging skin, lip, chin, forehead, nose, It is showing performance. However, 40-60% of the transplanted volume after fat transplantation has been reported that re-absorbed into the body (S Eremia et al, Dermatol Sur 2000, 26:..... 1150-1158; Fulton JE et al, Dermatol Clin , 2001, 19 (3): 523-530), fat skin transplant surgery in areas where skin is thin and subcutaneous fat is low causes problems such as cell clusters being touched, and the survival rate and survival time of transplanted fat are expected It is necessary to re-implant. Accordingly, there is a growing demand for a composition capable of sufficiently increasing the subcutaneous fat layer while replacing the fat grafting surgery with these problems.

Most of the cosmetic products that have the skin elasticity enhancing effect currently sold are those using common ingredients such as skin wrinkle removal or dermis strengthening. The lowering of skin elasticity is caused by degradation or destruction of collagen and elastin caused by aging or ultraviolet rays. Various materials have been developed and used for the purpose of inhibiting the reduction of collagen and elastin, which are the causes of such elasticity reduction. Among them, retinoids such as retinol and retinoic acid are typically used. Retinoids exhibit excellent resilience (Dermatology therapy 1998, 16, 357-364), but they suffer from the disadvantage that irritation occurs even when only a small amount is applied to the skin. Therefore, in order to solve the skin irritation problem of these raw materials, there is an increasing demand for the development of cosmetics using natural vegetable raw materials which are safer and have no irritation to the skin.

On the other hand, since the basic structure of the skin is maintained by subcutaneous adipose tissue, and the subcutaneous adipose tissue plays a role in the volume and intensity of the skin, as described above, the dermis or skin elasticity Increasing the volume of the adipose tissue is a better solution to maintain the volume and wrinkle of the skin than the method of giving, and recent articles related to this have been published (Kim WS et al., Sci. 2008, 34 (10): 1323-1326). [0095] In the present invention,

Studies using adipocytes have been actively conducted in various fields. A commonly used preadipocyte is a widely used cell because it has the property of differentiating into adipocytes by differentiation inducers such as insulin. Mesenchymal stem cells and adipocyte-derived stem cells are recently attracting attention in research using such adipocytes. Because these stem cells can be differentiated using methods similar to those used to differentiate adipose precursor cells into adipocytes in general (Xu Y et < RTI ID = 0.0 > al ., Exp. Cell Res. 2006, 312, 1856-1864) and is widely used in studies of adipocyte differentiation (Pittenger MF et al ., Science 1999, 284, 143-146).

Accordingly, the present inventors have made efforts to find herbal medicine extract promoting the differentiation of adipocytes constituting adipose tissue. As a result, it has been found that when the extract of soft-pit of flesh is treated on mesenchymal stem cells, mesenchymal stem cells effectively promote the differentiation process into adipocytes And completed the present invention.

Accordingly, it is an object of the present invention to provide a composition containing an herbal medicine extract that promotes differentiation of human mesenchymal stem cells into adipocytes, thereby increasing skin volume and elasticity and improving wrinkles.

In order to accomplish the above object, the present invention provides a composition for external application for skin and a pharmaceutical composition for promoting the differentiation of adipocytes, which comprises extract of pomegranate juice as an active ingredient.

The composition according to the present invention promotes the differentiation of mesenchymal stem cells into adipocytes by containing the extract of pomegranate as an active ingredient, so that it is useful for increasing the adipose tissue for cosmetic or cosmetic purposes. Thus, it increases skin volume and elasticity, It helps to improve.

FIG. 1 is a photograph showing the observation of a mesenchymal stem cell after inducing adipocyte differentiation after treatment of pine needle extract under an IDXT condition and then observing it with a microscope.
FIG. 2 is a graph showing the relative lipid differentiation level of mesenchymal stem cells after treatment of pine needle extract with respect to IDXT. FIG.

The present invention relates to a composition for promoting differentiation of adipocytes containing an extract of pome fruit as an active ingredient.

As used herein, the term "adipocyte differentiation" refers to the differentiation of adipose precursor cells or mesenchymal stem cells of an animal, preferably a mammal, into adipocytes, more preferably human mesenchymal stem cells, Is the differentiation of mesenchymal stem cells derived from human bone marrow into adipocytes.

Herbal medicine extracts used in the present invention are as follows.

Nelumbinis Semen is a mature seed of lotus, an aquatic plant that grows in reservoir-like ponds. It is mainly used for the fragile symptoms of the spleen to stop the diarrhea, and the heart beats the symptoms of insomnia. It also improves the kidney function and treats the fatigue and 夢 精. It treats the fatigue of the wife. The lower leaf, which is the lotus leaf, is used for summer diarrhea, headache and dizziness. Stop bleeding, nosebleeds, stool hemorrhage, uterine bleeding, and treat dizziness due to ejaculation after childbirth. The roots of lotus, the roots, are excellent in hemostatic effects such as blood, hematochezia, nosebleeds, fecal hemorrhage, hemorrhagic hemorrhage, urinary hemorrhage, and uterine hemorrhage. The federal, which is the overdose of the lotus, stops uterine bleeding, menorrhagia, and fecal bleeding, and is also used for hemorrhoids, hair loss, and acne. The stamina, which is the pistil of the lotus, has a lot of dreams, and it is used for the woolly, the well, the hemorrhage and the uterine bleeding, The green embryo in the seed is a lot of torment and anxiety, and it is used for gogal, oil well, hemorrhage, ocular hemorrhage. The pharmacological action was reported to suppress the nasal cancer and the throat. (Source, Naver Encyclopedia)

The pomegranate extract used in the present invention can be extracted by a conventional extraction method well known in the art using an organic solvent such as water or ethanol.

The composition of the present invention promotes the differentiation of Mesenchymal Stem Cell (MSC), thereby constituting a subcutaneous fat layer or providing an adipocyte to provide volume and elasticity to the skin and improve wrinkles, As an external preparation for skin or as a pharmaceutical composition.

When the composition of the present invention is used as a composition for external application for skin, it is not particularly limited in its formulation depending on the body part. Specifically, it may be a cosmetic composition having a formulation of a softening agent, a nutritional lotion, a massage cream, a nutritional cream, a pack, a gel or a skin adhesive type cosmetic composition and may be a cosmetic composition such as a lotion, ointment, gel, cream, patch or spray It may be a transdermal dosage form.

The composition for external application for skin according to the present invention contains an extract of pomegranate juice as an active ingredient in an amount of 0.0001 to 30% by weight based on the total weight of the composition for external application for skin.

In addition, in the composition for external application for skin according to each formulation, components other than the above-described extract of prawn extract of the present invention can 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, When applied at the same time, a synergistic effect can occur.

In addition, when the composition according to the present invention is used as a pharmaceutical composition, a pharmaceutical adjuvant such as a preservative, a stabilizer, a wetting agent or an emulsifying accelerator, a salt for controlling osmotic pressure and / or a buffer, And may be formulated into various oral or parenteral dosage forms according to conventional methods.

Examples of formulations for oral administration include tablets, pills, hard and soft capsules, liquids, suspensions, emulsions, syrups, granules, etc. These formulations may contain, in addition to the active ingredient, a diluent such as lactose, dextrose, Mannitol, sorbitol, cellulose and glycine) and lubricants (such as silica, talc, stearic acid and its magnesium or calcium salts and polyethylene glycols). The tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidine, optionally mixed with starch, agar, alginic acid or its sodium salt Such as disintegrants, absorbents, coloring agents, flavoring agents, and sweetening agents. Tablets can be prepared by conventional mixing, granulating or coating methods. Also typical of parenteral administration formulations are isotonic aqueous solutions or suspensions which are suitable for injectable use. 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 severity of the symptoms, the route of administration selected, the age, sex, weight and health of the subject, Are not intended to limit the scope of the invention in any way. In general, the dosage of herbal extracts 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 cosmetic or molding composition for promoting differentiation of mesenchymal stem cells according to the present invention includes all the adipose tissues of the body and is not limited to a specific site. For example, a breast, a hip Region. In addition, the effect of increasing the adipose tissue may be an 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. However, the following examples are intended to illustrate the present invention, but the scope of the present invention is not limited thereto.

[Reference Example 1] Preparation of crude drug extract

30 g of each of the broiler, control room, allium, and prawns were placed in 200 ml of ethanol (95.0 to 95.5 v / v%) and then pulverized for 20 minutes under a pressure of 1500 psi at 50 ° C using an ASE 300 ACCELERATED SOLVENT EXTRACOR (DIONEX Corporation). Further, it was further pulverized at 50 DEG C for 3 days using an ultrasonic washing machine (BRANSON Ultrasonics corporation). Each of the thus-pulverized extracts was dried at 40 ° C for 24 hours using a Modul spin 40 (Biotron corporation) apparatus to prepare samples of each extract.

[Test Example 1] Efficacy and quantitative measurement of promoting differentiation of human bone marrow-derived mesenchymal stem cells into adipocytes

Human bone marrow mesenchymal stem cells (hBM-MSCs) were purchased from Lonza, Inc. (Walkersville, MD, USA) and cultured according to Lonza's guidelines. Adipocyte differentiation was achieved using the method recommended by Ron, except that troglitazone was used instead of indomethacin to induce adipocyte differentiation of mesenchymal stem cells. Specifically, mesenchymal stem cells were cultured in the presence of insulin 1 μg / ml, dexamethasone (DEXA) 1 μM, isobutyl methyl xanthine (IBMX) 0.5 mM, and the stem cells were differentiated into adipocytes , And 10 ppm of seedlings were added to the medium under IDXT conditions to induce adipocyte differentiation. For comparison, the yolk, the control room and the alum extract were added to the medium at 10 ppm. As a positive control, troglitazone (TRO) 2 mM was used to treat with IDX medium. After 14 days from the start of adipocyte differentiation of mesenchymal stem cells, the cell culture medium was removed, and the cells were washed with 10 ml of phosphate buffered saline (PBS). Then, 0.2 ml / cm 2 of 10% formalin / PBS solution ² was used to fix the cells for 10 minutes. The fixed cells were washed once with a 60% isopropanol solution (0.5 ml / cm ² ), and the lipid in the adipocyte was stained with 0.2 ml / cm ² of a dye solution obtained by dissolving Oil Red O in 60% isopropanol Respectively. Subsequently, re-staining with hematoxylin was performed to easily distinguish nuclei, followed by observation with a microscope, and the result of treatment with the extract of soft cultured poultry was shown in Fig.

1, in the case of treatment with the extracts of the feces, the filtration chamber and the yeast extract, there was no significant difference from the degree of induction of adipocyte differentiation of the mesenchymal stem cells under the IDXT condition. On the other hand, Induced differentiation of adipocytes was more effective than induction of adipocyte differentiation of mesenchymal stem cells under IDXT conditions.

Then, the dyed oil red oi was eluted with a 100% isopropanol solution, and the absorbance was measured at a wavelength of 500 nm. The results are shown in Fig. At this time, each data value was corrected with an insulin control group. 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. In addition, in the case of treating each of the seeds extracts, the rate of promoting lipid differentiation relative to IDXT of mesenchymal stem cells is shown in Table 1 below.

A good name Scientific name IDXT contrast (growth rate) How Veratri Rhizoma 1% increase An operation room Ligustri Fructus 21% increase Abalone Forsythiae Fructus 27% increase Mature meat Nelumbinis Semen 75% increase

As can be seen from the results of Table 1 and FIG. 2, the extract of the prawn extract according to the present invention promotes differentiation of adipocytes without inducing apoptosis of mesenchymal stem cells, It can be confirmed that the differentiation of adipocytes can be promoted much more effectively than in the case of treatment.

[Test Example 2] Measuring effect of enhancing skin volume

To investigate the effect of the extract of the pine kernel of the present invention on the volume enhancement and wrinkle improvement of human skin, a questionnaire survey was conducted on 20 participants.

Specifically, a nutritional cream containing 2.0% by weight of the pomegranate extract as shown in Table 2 below was applied to the skin of the neck for 8 weeks once a day for one week. After that, And the effect of improvement was surveyed. When the effect is good, it is indicated by o, when it is normal, when it is not, when it is not, it is indicated by X. The results are shown in Table 3 below.

Compounding ingredient Content (% by weight) Purified water To 100 Pine nut extract 2.0 Vegetable hydrogenated oil 1.5 Stearic acid 0.6 Glycerol stearate 1.0 Cetearyl alcohol 2.0 Polyglyceryl-10pentastearate and behenyl alcohol and sodium stearoyl lactylate 1.0 Arachidyl behenyl alcohol 1.0 Cetylaryl alcohol & cetearyl glucoside 2.0 PEG-100 Stearate & Glycerololate & Propylene Glycol 1.5 Caprylic / Carrick Triglyceride 11.0 Cyclomedicone 6.0 Preservative, incense Suitable amount Triethanolamine 0.1

A good name Volume enhancement effect (persons) ○ △ X How 2 2 16 An operation room 4 5 11 Abalone 5 4 11 Mature meat 15 3 2

As can be seen from the above Table 3, the skin volume and wrinkle improving effect are not good when the feces, the labial room, and the articulation are respectively used. However, when the composition containing the extract of the flesh of the present invention is used, It can be confirmed that the effect of improving skin volume and wrinkle is good.

From the results of the above experiment, when the composition containing the extract of green tea and lard is applied to the facial skin and invaded or taken into the skin, the effective ingredient of the extract increases the volume of fat tissue, thereby increasing the volume and elasticity of the skin, It can be understood that the effect of improving the performance can be obtained.

Examples of formulations for the composition of the present invention are illustrated below.

[Formulation Example 1] Tablets

100 mg of the extract of one kind, 400 mg of lactose, 400 mg of corn starch and 2 mg of magnesium stearate were mixed in the pine meat according to the present invention, followed by tableting according to a conventional preparation method.

[Formulation Example 2]

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

Claims (11)

delete delete delete delete delete delete A method for differentiating mesenchymal stem cells into adipocytes under in vitro conditions using a composition containing extracts of pomegranate juice as an active ingredient. [8] The method according to claim 7, wherein the mesenchymal stem cells are human mesenchymal stem cells. 9. The method according to claim 8, wherein the human mesenchymal stem cell is a mesenchymal stem cell of a human bone marrow. [Claim 8] The method according to claim 7, wherein the pomegranate extract is an extract obtained by extracting water, ethanol, or a mixed solvent of water and ethanol with an extraction solvent. 11. The method according to claim 10, wherein the concentration of ethanol in the water-ethanol mixed solvent is 95% or more.

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