KR20120026689A - Composition for promoting the differentiation of human mesenchymal stem cell - Google Patents

Abstract

PURPOSE: A composition for promoting human mesenchymal stem cell differentiation is provided to enhance skin volume and elasticity and to improve anti-wrinkling. CONSTITUTION: An external use skin composition for promoting adipocytes contains 0.0001-30 wt% of Artemisia asiatica extract as an active ingredient. The composition promotes differentiation of mesenchymal stem cells into adipocytes. The mesenchymal stem cells are derived from human body. A pharmaceutical composition for promoting differentiation into adipocytes contains the extract as an active ingredient. The dose of the extract is 0.001 mg/kg/day-2,000 mg/kg/day.

Description

Composition for promoting the differentiation of human mesenchymal stem cell}

The present invention relates to a composition for promoting the differentiation of human mesenchymal stem cells, and more particularly, by containing the leaf extract as an active ingredient to promote the differentiation of human mesenchymal stem cells into adipocytes (Lipid droplet) The present invention relates to a composition for promoting differentiation of human mesenchymal stem cells, which promotes volume and elasticity of skin and improves wrinkles.

The skin is the largest organ of the human body, occupying about 16% of our body volume, 1.8 m², 2-4 mm thick, and weighing about 3 kg. The epidermis, dermis and subcutaneous fat layer. It consists of. The skin is in direct contact with the external environment and serves as an important protective film to protect the human body from various harmful factors such as human harmful microorganisms and external stimuli such as ultraviolet rays, while maintaining the biochemical functions necessary for metabolism of the whole body. It is an indispensable organ. In particular, the subcutaneous fat layer under the dermis is composed of the lobules of adipocytes, which act as a cushion to absorb and protect the internal organs of the skin against external heat and nourish the triglycerides. It is also stored in organelles called lipid droplets and used as an energy source when needed. However, with age, the skin is damaged by natural factors and various external stimuli. In particular, the activity and expression of collagen and elastic fiber (elastin), which are responsible for elasticity in the dermis, are reduced and elasticity is reduced. At the same time, in the subcutaneous fat, the production of fat decreases, and eventually, the constituent cells of the entire skin do not function properly, resulting in skin aging such as wrinkles caused by loss of elasticity.

In the modern world, especially for women, skin is a major indicator of their health, so skin that loses its elasticity as it ages becomes a sensitive issue for women. In particular, the skin on the face is thinner than other skin areas, and it is easy to sag and lose elasticity due to factors such as weight and age. In addition, the decrease in facial elasticity and fat caused by metabolic abnormalities such as hormones, blood, fat, and bone, which occur mainly in postmenopausal women, make women feel a great loss of femininity. Increasing the volume of adipose tissue is a solution to the problems that are very sensitive to women in modern society, and many women try to maintain or increase the firmness of the skin through plastic surgery with side effects and economic burden. do.

Currently, a widely used method for improving the decrease in elasticity of the skin is liposuction. Lipograft surgery is a procedure in which a tulip needle-shaped needle is taken using a syringe having a needle that is not sharp and transplanted to each part of the skin where elasticity is poor. These fat grafts can be applied to fill and improve the wrinkles of aging skin as it ages, to correct the lip, jaw, and forehead lines, skin impregnated by nose shaping and burns or wounds, and areas lost due to cancer resection. The results are showing. However, it has been reported that 40-60% of the graft volume is absorbed back into the body after liposuction (S Eremia et al., Dermatol . Sur. 2000, 26: 1150-1158; Fulton JE et al., Dermatol . Clin 2001, 19 (3): 523-530), fat transplantation of skin is thin subcutaneous fat area is less raised a problem of which the cell mass touched, actually the transplanted fat survival and survival expectancy There is a case in which replanting is not necessary. Therefore, there is an increasing demand for a composition that can sufficiently increase the subcutaneous fat layer while replacing lipostomy with these problems.

Most of the cosmetics that have a skin elasticity promoting effect currently on the market using ingredients having the general efficacy, such as skin wrinkle removal or strengthening the dermis. Decreased skin elasticity is caused by decreased production or destruction of collagen and elastin caused by aging or ultraviolet rays. Various substances have been developed and used for the purpose of suppressing the reduction of collagen and elastin, which causes the decrease in elasticity. Among them, retinoids such as retinol and retinoic acid are typically used. Retinoids have an excellent elasticity-improving effect (Dermatology therapy 1998, 16, 357-364), but they have the disadvantage that irritation occurs even when only a small amount is applied to the skin. Therefore, in recent years, in order to solve the skin irritation problem of these raw materials, there is a high demand for the development of cosmetics using natural vegetable raw materials that are safer and have no skin irritation.

Meanwhile, since the basic structure of the skin is maintained by the subcutaneous fat tissue, and the subcutaneous fat tissue plays a role in determining the volume and strength of the skin, elasticity is applied to the dermis or epidermis of the outer skin layer that has been used as described above. Increasing the volume of adipose tissue rather than imparting it may be a good solution for maintaining the volume of the skin and improving wrinkles, and recent papers have been published (Kim WS et. al ., J. Dermatol. Sci . 2009, 53 (2): 96-102; Trottier V et al ., Stem Cells . 2008 26 (10): 2713-23; Park BS et al ., Dermatol . Surg . 　 2008, 34 (10): 1323-1326).

Research using fat cells has been actively conducted in various fields. Preadipocytes generally used are widely used cells because they have the property of differentiating into adipocytes by differentiation inducing substances such as insulin. Recently, attention has been drawn in the study using the adipocytes, such as mesenchymal stem cells (adipocyte derived stem cells). These stem cells can generally be differentiated using methods similar to those used to differentiate adipocytes into adipocytes (Xu Y et. al ., Exp. Cell Res. 2006, 312, 1856-1864), widely used in the study of adipocyte differentiation (Pittenger MF et al ., Science 1999, 284, 143-146).

Therefore, the present inventors have tried to find a herbal extract that promotes the differentiation of fat cells that make up adipose tissue, and found that when mesenchymal extracts were treated on mesenchymal stem cells, mesenchymal stem cells effectively promoted the process of differentiating into adipocytes. This invention was completed.

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

In order to achieve the above object, the present invention provides a skin external preparation composition and pharmaceutical composition for promoting adipocyte differentiation containing the leaf extract as an active ingredient.

The composition according to the present invention is useful for increasing adipose tissue for cosmetic or cosmetic purposes by promoting the differentiation of mesenchymal stem cells into adipocytes by containing the leaf extract as an active ingredient, thus increasing skin volume and elasticity and reducing skin wrinkles. It is helpful to improve.

Figure 1 is a photograph showing the observation under a microscope after inducing adipocyte differentiation of mesenchymal stem cells by treating the mesenchymal extract under IDXT conditions.
Figure 2 is a graph showing the relative fat differentiation value compared to IDXT of mesenchymal stem cells after treatment of the leaf lobe extract.

The present invention relates to a composition for promoting adipocyte differentiation comprising a leaf extract as an active ingredient.

As used herein, the term “fat cell differentiation” refers to the differentiation of adipocytes or mesenchymal stem cells from animals, preferably mammals into adipocytes, and more preferably human mesenchymal stem cells. Preferably, mesenchymal stem cells derived from human bone marrow differentiate into adipocytes.

The herbal extract used in the present invention is as follows.

The appearance of Artemisiae Argyi Folium is composed of pressed leaves and pieces, often containing thin stems. The leaves are crooked, but when they are unfolded, they are divided into feathers and attached alternately. The upper side is green or dark greenish brown, and the back side is dense white and gray downy hair, giving a soft touch. The pharmacological action is to warm the blood vessels (氣血) and the meridians (經脈), so the uterine and lower abdomen, weak uterine bleeding, bleeding during pregnancy, hemostasis, hemostasis, nosebleeds and hemostasis. In addition, it is weak and cold in the lower part of the stomach, cold and pain in the abdomen, dysmenorrhea, dysmenorrhea and soybeans (등에 下) used in the eczema and skin itching is effective. In addition, there are hemostatic action, fungal action, bronchial smooth muscle relaxation action, antitussive expectoration action, sleep action, uterus excitement action and hypersensitivity shock protection.

The leaf extract used in the present invention can be extracted by conventional extraction methods 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 Cells (MSCs), thereby constituting the subcutaneous fat layer or providing fat cells to provide the effect of providing volume and elasticity to the skin and improving wrinkles. Or it may be a molding composition, it may be provided as a topical skin composition or a pharmaceutical composition.

When the composition according to the present invention is used as a skin external preparation composition, it is not particularly limited in the formulation according to the body part. Specifically, for example, it may be a cosmetic composition having a formulation of a softening lotion, nourishing lotion, massage cream, nutrition cream, pack, gel or skin adhesive type cosmetics, and may also be a lotion, ointment, gel, cream, patch or spray Transdermal dosage forms.

The skin external preparation composition according to the present invention contains the leaf extract in an amount of 0.0001-30% by weight based on the total weight of the composition.

In addition, in the composition for external application for skin according to each formulation, other components other than the above-mentioned leaf extract of the present invention can be appropriately selected and blended by those skilled in the art without difficulty according to the formulation or use purpose of other external preparation for skin, in which case, At the same time, synergistic effects may occur.

In addition, when the composition according to the present invention is used as a pharmaceutical composition, it further contains a pharmaceutical adjuvant such as preservatives, stabilizers, hydrating or emulsifying accelerators, salts and / or buffers for controlling osmotic pressure and other therapeutically useful substances. And may be formulated in various oral or parenteral dosage forms according to conventional methods.

Formulations for oral administration include, for example, tablets, pills, hard and soft capsules, solutions, suspensions, emulsifiers, syrups or granules. These formulations may contain, in addition to the active ingredients, diluents (e.g., lactose, dextrose, sucrose, Mannitol, sorbitol, cellulose and glycine) and glidants such as silica, talc, stearic acid and its magnesium or calcium salts and polyethylene glycols. Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidine, optionally starch, agar, alginic acid or its sodium salt It may contain pharmaceutical additives such as disintegrants, absorbents, colorants, flavors and sweeteners. Tablets may be prepared by conventional mixing, granulating or coating methods. Also representative of parenteral formulations are injectable formulations, preferably aqueous isotonic solutions or suspensions.

It is to be understood that the actual dosage of the active ingredient should be determined in light of several relevant factors such as the severity of the symptom, the route of administration chosen, the age, sex, weight and health of the subject. Therefore, the dosage of said leaf | leaf extract does not limit the scope of the present invention by any method. Generally, the dosage of the mesenchymal extract ranges from 0.001 mg / kg / day to approximately 2,000 mg / kg / day. More preferred dosages are from 0.5 mg / kg / day to 2.5 mg / kg / day.

The site of application of the composition for promoting differentiation of mesenchymal stem cells according to the present invention includes all of the adipose tissue of the body, but is not limited to a specific site, for example, it may include the breast and hip areas of women with a lot of fat layers. Can be. In addition, the increasing effect of the adipose tissue, but the effect of increasing the adipose tissue for cosmetic or cosmetic purposes, but is not limited thereto.

Hereinafter, the present invention will be described more specifically with reference to examples and test examples. These examples are provided only for understanding the contents of the present invention, and the scope of the present invention is not limited to these examples, and modifications, substitutions, and insertions commonly known in the art may be performed. This is also included in the scope of the present invention.

Reference Example 1 Preparation of Herbal Medicine Extract

Each 30 g of flax, benzoate and leaf buds were placed in 200 ml of ethanol (95.0-95.5 v / v%) and then ground for 20 minutes at 1,500 psi pressure at 50 ° C. using an ASE300 ACCELERATED SOLVENT EXTRACOR (DIONEX Corporation) instrument. In addition, using an ultrasonic cleaner (BRANSON Ultrasonics corporation) sonicator device was further ground for 3 days at 50 ℃. Each of the pulverized extracts were dried at 40 ° C. for 24 hours using a module spin 40 (Modul spin 40; Biotron corporation) instrument to prepare each extract sample.

Experimental Example 1 Efficacy and Quantitative Measurement 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 instructions. Adipocyte differentiation was achieved using the method recommended by Lonza, except that Troglitazone was used instead of indomethacin to induce adipocyte differentiation of mesenchymal stem cells. Specifically, 1 μg / ml of insulin, 1 μM of dexamethasone (DEXA: Dexamethasone) and 0.5 mM of isobutyl methyl xanthine (IBMX: isobutyl methyl xanthine) were added to culture medium of the mesenchymal stem cells to differentiate stem cells into adipocytes. (Hereinafter, referred to as "IDXT"), 10 ppm of leaflets, linseed and benzoin extracts were added to each medium under IDXT conditions at the time of inducing adipocyte differentiation. As a positive control, Troglitazone (TRO) 2 mM was used and treated with IDX medium (insulin, DEXA and IBMX treated medium).

After 14 days of adipocyte differentiation of mesenchymal stem cells, the cell culture medium was removed, the cells were washed with 10 ml of phosphate buffered saline (PBS), and then 0.2 ml / cm 2 of 10% formalin / PBS solution. Cells were fixed for 10 minutes. The fixed cells were washed once with 0.5 ml / cm 2 of 60% isopropanol solution, and lipid bodies were stained for 10 minutes with 0.2 ml / cm 2 staining solution in which Oil Red O was dissolved in 60% isopropanol. Subsequently, restaining with hematoxylin was performed to make cell nuclei easily distinguished, and then the results of the observation under a microscope are shown in FIG. 1.

Referring to Figure 1, the treatment of the flax extract and benzoin extract was little different from the degree of inducing adipocyte differentiation of mesenchymal stem cells under IDXT conditions, but under the IDXT conditions in the medium added with the lobe extract under IDXT conditions It was confirmed that the differentiation of adipocytes occurs more effectively than the induction of adipocyte differentiation of mesenchymal stem cells.

Then, after dyeing the oil red o was dissolved in 100% isopropanol solution, 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. This increase was statistically significant, and the statistics were verified by the two-tailed Student's t test, indicating that there was a significant difference when the p value was less than 0.05. In addition, the growth rate of fat differentiation compared to IDXT (negative control group) of mesenchymal stem cells in the medium treated with each test substance is shown in Table 1 below.

Herbal medicine Scientific name IDXT vs. Growth Rate Flax Lini Semen 4% reduction Benzoin Enzoinum 20% increase Love Artemisiae Argyi Folium 36% increase

As can be seen from the results of Table 1 and Figure 2, the leaf lobe extract according to the present invention promoted the differentiation of adipocytes without inducing cell death of mesenchymal stem cells, respectively, Compared with the case, it was confirmed that it can significantly promote the differentiation of adipocytes.

[Formulation Example 1 and Comparative Formulation Examples 1, 2]

The nutritional creams of Formulation Example 1 and Comparative Formulation Examples 1 and 2 were prepared in a conventional manner according to the compositions shown in Table 2 below (unit: wt%).

Compounding ingredient Formulation Example 1 Comparative Formulation Example 1 Comparative Formulation Example 2 Purified water Balance Balance Balance Leaf extract 2.0 - - Flax Extract - 2.0 - Benzoin Extract - - 2.0 Vegetable hydrogenated oil 1.5 1.5 1.5 Stearic acid 0.6 0.6 0.6 Glycerol stearate 1.0 1.0 1.0 Cetearyl alcohol 2.0 2.0 2.0 Polyglyceryl-10 Pentastearate & Behenyl Alcohol & Sodium Stearoyl Lactylate 1.0 1.0 1.0 Arachidyl behenyl alcohol 1.0 1.0 1.0 Cetylaryl alcohol & cetearyl glucoside 2.0 2.0 2.0 PEG-100 Stearate & Glycerololate & Propylene Glycol 1.5 1.5 1.5 Caprylic / Carlic Triglycerides 11.0 11.0 11.0 Cyclomedicon 6.0 6.0 6.0 Preservative, incense Quantity Quantity Quantity Triethanolamine 0.1 0.1 0.1

Test Example 2 Measurement of Skin Volume Enhancement Effect

In order to investigate the effect of the nutrition creams prepared in Formulation Example 1 and Comparative Formulation Examples 1 and 2 on the improvement of human skin volume and wrinkle improvement, a questionnaire was conducted among 20 clinical trial participants.

Participants were asked to apply the nourishing creams of Formulation Example 1 and Comparative Formulation Examples 1 and 2 once a day to the skin of the neck area for 8 weeks. When the effect is good ○, the normal case is △, when there is no effect is represented by X, the results are shown in Table 3 below.

Herbal medicine Volume improvement effect (the number of the respondents) ○ △ X Formulation Example 1 14 3 3 Comparative Formulation Example 1 One 5 14 Comparative Formulation Example 2 7 6 7

In the results of Table 3, when Comparative Examples 1 and 2 including flax and benzoin were used, the effect of improving skin volume and wrinkles was not good, but when Formulation Example 1 containing the leaf extract according to the present invention was used. It was confirmed that most test participants judged that skin volume enhancement and wrinkle improvement were good.

From the results of the experiment, each of the compositions containing the leaf extracts applied to the skin of the face, invading or taking the skin, the active ingredient of the extract increases the volume of adipose tissue, thus increasing the volume and elasticity of the skin and reduce skin wrinkles It can be seen that the effect of improving can be obtained.

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

Preparation Example 1 Tablet

100 mg of leaf extracts, 400 mg of lactose, 400 mg of corn starch, and 2 mg of magnesium stearate were mixed, and then tableted according to a conventional method for preparing tablets.

Preparation Example 2 Capsule

100 mg of leaf extracts, 400 mg of lactose, 400 mg of corn starch, and 2 mg of magnesium stearate were mixed, and then capsules were prepared by filling gelatin capsules according to a conventional method for preparing capsules.

Claims (11)

Skin external preparation composition for promoting the differentiation of fat cells containing the leaf extract as an active ingredient. The composition for external application for skin according to claim 1, wherein the composition is for promoting the differentiation of mesenchymal stem cells into adipocytes. The composition for external application of skin according to claim 2, wherein the mesenchymal stem cells are human mesenchymal stem cells. The composition for applying the external of the skin according to claim 2, wherein the composition is for increasing adipose tissue. According to claim 1, wherein the composition is a topical skin composition, characterized in that it contains 0.0001 to 30% by weight relative to the total weight of the composition. The external preparation composition for skin according to claim 1, wherein the composition is for increasing skin volume. The composition for applying the external of the skin according to claim 1, wherein the composition is for increasing skin elasticity. The composition for applying the external of the skin according to claim 1, wherein the composition is for improving skin wrinkles. A pharmaceutical composition for promoting adipocyte differentiation containing the leaf extract as an active ingredient. The pharmaceutical composition of claim 9, wherein the composition is intended to promote differentiation of mesenchymal stem cells into adipocytes. 10. The pharmaceutical composition of claim 9, wherein the dosage of the leaf extract is 0.001 mg / kg / day to 2,000 mg / kg / day.

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