KR20120140450A - Composition for promoting the differentiation of human mesenchymal stem cell - Google Patents

Abstract

PURPOSE: A composition with Nelumbinis Semen extracts as an active ingredient for promoting the differentiation of adipocyte in human mesenchymal stem cell is provided to promote differentiating the adipocyte from a human mesenchymal stem cell to produce lipid droplet, enhance the electricity and the volume of skin and reduce wrinkles. CONSTITUTION: A composition for skin external application for promoting the differentiation of adipocyte contains Nelumbinis Semen extracts as an active ingredient. The composition promotes differentiating adipocyte from a mesenchymal stem cell. The mesenchymal stem cell is a human mesenchymal cell. The composition increases adipocyte tissue. For the total weight of the composition, a Nelumbinis Semen extract weighs ranging from 0.001 to 30%. The composition enhances the volume of skin and the elasticity of skin, and reduces skin wrinkles. A pharmaceutical composition for promoting the differentiation of a adipocyte cell contains Nelumbinis Semen as an active ingredient. A Nelumbinis Semen extract is administered from 0.001mg /kg /day-2000mg /kg /day. [Reference numerals] (AA) Relative revelation amount; (BB) Negative control group; (CC) Ligustri Fructus; (DD) Veratri Rhizoma Et Radix; (EE) Forsythiae Fructus; (FF) Nelumbo semen

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 into adipocytes, and more particularly, by containing the extract of the soft muscle meat as an active ingredient to promote differentiation of human mesenchymal stem cells into adipocytes. Lipid droplets) to improve the volume and elasticity of the skin and to improve the wrinkles.

The subcutaneous fat layer under the dermis is composed of the lobules of adipocytes, which act as a cushion to absorb and protect the organs inside the skin against external heat, and the nutrients form triglycerides. They are stored in organelles called lipid droplets and used as energy sources 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 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, 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. 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.

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 applying 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., Sci. 2009, 53 (2). ): 96-102; Throtttier V et al., Stem cells. 2008 26 (10): 2713-23; Park Bs et al., Dermatol. Surg. 2008, 34 (10): 1323-1326).

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. 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 the adipose tissue, and found that the mesenchymal stem cells effectively promote the process of differentiating mesenchymal stem cells into adipocytes when the extracts were treated on mesenchymal stem cells. This invention was completed.

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 achieve the above object, the present invention provides a skin external preparation composition and pharmaceutical composition for promoting adipocyte differentiation containing the extract of lotus root meat 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 extract of lotus root 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 induction of adipocyte differentiation of mesenchymal stem cells after treatment of the extract of the lotus leaf under IDXT conditions.
Figure 2 is a graph showing the relative fat differentiation value compared to IDXT of mesenchymal stem cells after the treatment of the extract of the lotus leaf.

The present invention relates to a composition for promoting adipocyte differentiation containing an extract of lotus root meat as an active ingredient.

As used herein, the term "fat cell differentiation" refers to the differentiation of adipocytes or mesenchymal stem cells of an animal, preferably a mammal, 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.

Nelumbinis Semen is a mature seed of lotus, aquatic plants that grow in ponds like reservoirs. The spleen is mainly used for fragile symptoms to stop diarrhea, and the heart beats and treats the symptoms of insomnia. In addition, by increasing the function of the kidneys (유 精) and dreams (치료 精) to treat the wife's white crabs. The lower leaves, the leaves of lotus, are used for summer diarrhea, headaches and dizziness. Stop blood, nosebleeds, stool bleeding, uterine bleeding, and cure dizziness caused by bleeding after giving birth. The root of lotus, the root, has excellent hemostatic effect on keratin, hemostasis, nosebleed, fecal bleeding, hemorrhoidal bleeding, urine bleeding, and uterine bleeding. The Commonwealth of Lotus, which stops uterine bleeding, excessive menstruation and stool bleeding, is also used for hemorrhoids, prolapse and swelling. Yeonsu, a pistil of lotus, has many dreams, and is used for dreams, oil wells, bleeding and uterine bleeding, crayfish, and dysentery. Yeonsimsim, a green embryo in seeds, has a lot of anxiety, anxiety, shame, and it is used for dry mouth, oil well, hemostasis, and eye bleeding. Pharmacological action has been reported to inhibit nasal cancer and throat. (Source, Naver Encyclopedia)

Soft lotus 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 Cell (MSC), thereby constituting a subcutaneous fat layer or providing an adipocyte to provide volume and elasticity to 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 external preparation for skin composition according to the present invention contains the extract of the soft lotus meat as an active ingredient in an amount of 0.0001 to 30% by weight based on the total weight of the external preparation for skin composition.

In addition, in the composition for external application for skin according to each formulation, other ingredients other than the soft extract of the present invention described above may 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, 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 may be prepared by conventional mixing, granulating or coating methods. Also representative of parenteral formulations are injectable formulations, preferably aqueous isotonic solutions or suspensions. However, it should 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 subject's age, sex, weight and health condition, and therefore the dosage of the herbal extracts. Does not limit the scope of the invention in any way. Generally, the dosage of the herbal extracts ranges from 0.001 mg / kg / day to approximately 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 only for illustrating the present invention, and the scope of the present invention is not limited thereto.

[Reference Example 1] Preparation of crude drug extract

For example, 30 g each of the journey room, the pontoon, and the soft meat were put in 200 ml of ethanol (95.0 to 99.5v / v%), and then pulverized for 20 minutes at 1500 ° C. under a pressure of 1500 psi at 50 ° C. using an ASE300 ACCELERATED SOLVENT EXTRACOR (DIONEX Corporation) instrument. 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, 1 μg / ml of insulin, 1 μM of dexamethasone (DEXA) and 0.5 mM of isobutyl methyl xanthine (IBMX) were cultured in the culture of mesenchymal stem cells to differentiate stem cells into adipocytes. At the time of inducing adipocyte differentiation, 10 ppm each of the extracts of the lotus root meat was added to the medium under IDXT conditions. In addition, for comparison, Journey, Journey, and Fructus extracts were added to the medium by 10 ppm. As a positive control, Troglitazone (TRO) 2 mM was used and treated with IDX medium. After 14 days of adipocyte differentiation of mesenchymal stem cells, the cell culture was removed, the cells were washed with 10 ml of Phosphate Buffered Saline (PBS), and then 0.2 ml / cm of 10% formalin / PBS solution. Cells were fixed for 10 minutes using ² . The fixed cells were washed once with 0.5 ml / cm ² of 60% isopropanol solution, and stained with lipids in adipocytes with 0.2 ml / cm ² stained with Oil Red O in 60% isopropanol for 10 minutes. It was. Subsequently, re-staining with hematoxylin (hematoxylin) was performed to make cell nuclei easily distinguished, and then observed under a microscope.

Referring to Figure 1, the case of treatment of the yeast, Journey, Yeongyo extract was not significantly different from the degree of inducing adipocyte differentiation of mesenchymal stem cells under IDXT conditions, whereas mesenchymal stem was added after the addition of the extract of lotus leaf muscle under IDXT conditions. Induction of adipocyte differentiation of cells can be confirmed that the differentiation of adipocytes occurs more effectively 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, the increase in fat differentiation promotion compared to IDXT of mesenchymal stem cells, respectively, when treated with the extract of the soft muscle is shown in Table 1 below.

Herbal medicine Scientific name IDXT vs. Growth Rate Way Veratri Rhizoma 1% increase Journey Room Ligustri Fructus 21% increase Abalone Forsythiae Fructus 27% increase Lotus root Nelumbinis Semen 75% increase

As can be seen from the results of Table 1 and Figure 2, the extracts of the lotus leaf muscle according to the present invention promotes the differentiation of adipocytes without inducing cell death of mesenchymal stem cells, respectively, and the extracts of the yeast, journey, and ducts, respectively It can be seen that it can promote the differentiation of adipocytes much more effectively than when treated.

[Test Example 2] Measuring effect of enhancing skin volume

In order to find out the effect of improving the volume and wrinkles of human skin when using the extract of the present invention, a questionnaire was conducted among 20 clinical trial participants.

Specifically, the nourishing cream containing the extract of lotus root meat as shown in Table 2 in an amount of 2.0% by weight based on the total weight of the composition was applied to the skin of the neck area once a day for 8 weeks, after which volume enhancement and wrinkles were applied. A survey was conducted on the effectiveness of the improvements. 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 Lotus root extract 2.0 Vegetable hydrogenated oil 1.5 Stearic acid 0.6 Glycerol stearate 1.0 Cetearyl alcohol 2.0 Polyglyceryl-10 Pentastearate & Behenyl Alcohol & 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 / Carlic Triglycerides 11.0 Cyclomedicon 6.0 Preservative, incense Suitable amount Triethanolamine 0.1

Herbal medicine Volume enhancement effect (persons) ○ △ X Way 2 2 16 Journey Room 4 5 11 Abalone 5 4 11 Lotus root 15 3 2

As can be seen in Table 3 above, the use of the route, the journey room, and the bridge is not good for improving the skin volume and wrinkles, while using the composition containing the extract of the present invention, most of the test participants It can be seen that skin volume enhancement and wrinkle improvement are good.

From the results of the experiment, when each of the compositions containing the extract of lotus root meat is applied to the face skin, 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 reducing the 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

Tablets were prepared by mixing 100 mg of one extract, 400 mg of lactose, 400 mg of corn starch, and 2 mg of magnesium stearate in tablets according to the present invention.

[Formulation Example 2]

In the lotus root according to the present invention, 100 mg of one extract, 400 mg of lactose, 400 mg of corn starch, and 2 mg of magnesium stearate were mixed, followed by filling into a gelatin capsule according to a conventional method for preparing a capsule to prepare a capsule.

Claims (11)

Skin external preparation composition for promoting the differentiation of fat cells containing lotus root extract as an active ingredient. The composition for external application for skin according to claim 1, wherein the composition promotes 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. 3. The composition for external application for skin according to claim 2, wherein the composition increases fat tissue. The composition for applying the external of the skin according to claim 1, wherein the composition is contained in an amount of 0.0001 to 30% by weight based on the total weight of the lotus root extract composition. The composition for external application for skin according to claim 1, wherein the composition increases skin volume. The external preparation composition for skin according to claim 1, wherein the composition increases skin elasticity. The composition for external application for skin according to claim 1, wherein the composition improves skin wrinkles. A pharmaceutical composition for promoting the differentiation of fat cells containing the extract of lotus root as an active ingredient. 10. The pharmaceutical composition according to claim 9, wherein the composition promotes the differentiation of mesenchymal stem cells into adipocytes. The pharmaceutical composition according to claim 9, wherein the composition has a dose of 0.001 mg / kg / day to 2000 mg / kg / day.

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