KR20170025205A - Composition for Facilitating the Proliferation of Stem Cells Comprising the Extract of Angelicae Dahuricae Radix as an Effective Ingredient - Google Patents

Abstract

The present invention provides a composition for stimulating proliferation of stem cells, which comprises a white-ground extract as an active ingredient. The present invention provides a composition for promoting stem cell proliferation, It can be usefully used when it is necessary to prepare or supply a sufficient amount of stem cells before being differentiated into a specific tissue within a short period of time.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for promoting stem cell proliferation comprising an extract of a blanched paper as an active ingredient,

The present invention relates to a composition for promoting proliferation of stem cells and a method for promoting proliferation of stem cells using the same.

Recently, the use of stem cells in the field of regenerative medicine using tissue engineering has been proposed as a new field for the treatment of incurable diseases. Therefore, interest in stem cell research has increased, and it has been recognized that stem cells capable of forming a tissue through proliferation and differentiation can solve not only most diseases but also tissue damage.

Stem cells have the ability to self-replicate under undifferentiated conditions and differentiate into specific cells under appropriate conditions. Stem cells can be divided into embryonic stem cells and adult stem cells depending on their origins. Since human embryonic stem cells are derived from embryos that can occur in human life forms, they have excellent cell proliferation and differentiation potential, but have bioethical problems. On the other hand, adult stem cells have a limited ability to differentiate from embryonic stem cells, but they have been developed into stem cells by collecting cells already existing in various organs of the human body from bone marrow, blood, brain, and skin, . As adult stem cells, there are hematopoietic stem cells, mesenchymal stem cells, neuroblastoma, epithelial cells, etc. Hematopoietic stem cells can produce hematopoietic cells and lymphocytes, and are useful for the treatment of immune system diseases including hematologic malignancies. Stem cells are differentiated into bone, cartilage, fat, and fibrous tissue, and are involved in the recovery of each tissue when damaged. However, these adult stem cells are present in small amounts in each tissue and may exist for many years without division or proliferation.

In other words, the term "stem cell" refers to a "undifferentiated" cell that has this differentiation ability but does not yet undergo differentiation. In this undifferentiated state, stem cells can be differentiated into various tissue cells by appropriately matching the conditions, and studies on the treatment of diseases using such cells have been actively conducted. However, as ethical problems of embryonic stem cells have emerged, Studies have been focused on using adult stem cells with fewer problems.

Since stem cells obtained from tissues are small in quantity and require a large amount of cells in use, culturing is performed to increase the number of stem cells. For example, in Patent Document 1, stem cells are proliferated in vitro using a bioreactor Method is disclosed. However, research on the culture composition capable of promoting the proliferation of stem cells while maintaining the characteristics of the stem cells when culturing for stem cell proliferation is somewhat lacking.

On the other hand, the herbivorous plant belongs to the perennial herbaceous plant, which is a perennial herbaceous plant, and has been used as edible and medicinal plants, including Angelica japonica, Angelica japonica, Angelica gigas, Gibbons and Giblets. Among them, Guri is a 2-3-year-old herbarium native to Korea, China, and Japan. The scientific name of Guri is Angelica dahurica BENTHAM et HOOKER. There are many genera, ), Copper stand, and goji medium. It is 1-2 m in height, 7-8 cm in diameter at the bottom, has fine hairs on the upper part, branches are split and roots are thick. It is said that the roots of this species and its variants are called white pheasant. White pheasant has been known to be effective for skin pruritus, headache, calmness, seizure, hemostasis, facial pain, facial neuralgia,

Therefore, the present inventors have studied a method of increasing the proliferation while maintaining stem cell characteristics, and have found that when Angelicae dahuricae radix extract is cultured in the culture of gingival stem cells, the proliferation degree is increased The present invention has been completed.

Korean Patent Publication No. 10-2009-0021329

An object of the present invention is to provide a composition for promoting stem cell proliferation using an effective ingredient derived from a root of a plant belonging to the genus Bacillus, and a method for promoting the proliferation of stem cells using the composition.

In order to accomplish the above object, one aspect of the present invention provides a composition for promoting stem cell proliferation comprising a blanched extract.

According to another aspect of the present invention, there is provided a method for promoting stem cell proliferation comprising the step of treating a stem cell with a blanched extract.

The composition for stimulating proliferation of stem cells according to the present invention has the effect of promoting the proliferation of the stem cells themselves while maintaining the characteristics of the stem cells, so that the stem cells before differentiation into specific tissues can be prepared or supplied in a sufficient amount in a short period of time It may be usefully used in cases such as when it is necessary.

However, the effects of the present invention are not limited to the above-mentioned effects, and other effects not mentioned can be clearly understood by those skilled in the art from the following description.

FIGS. 1 to 3 are photographs showing changes in cell morphology at 1 day, 3 days, and 7 days after treatment with adult stem cells derived from gill.
FIGS. 4 and 5 are graphs showing the results of measurement of the degree of cell proliferation at 1 day and 7 days after treatment of adult stem cells derived from gill, respectively, on the white bark extract.

First, terms used in the specification of the present invention will be described.

The term " stem cell " referred to in the present invention refers to a cell having pluripotent or totipotent self-renewal capable of differentiating into cells of all tissues of the individual Cells, and includes embryonic stem cells, inducible pluripotent stem cells, and adult stem cells.

The term " embryonic stem cell " refers to a cell obtained by extracting an inner cell mass from a blastocyst embryo immediately before fertilization of the embryo into the uterus of a mother, cultivating the same in vitro, (Pluripotent) or omnipotent (totipotent) self-renewal stem cells (self-renewal) refers to stem cells.

The term ' inducible pluripotent stem cells ' refers to cells that have been induced so that the differentiated cells have pluripotent differentiation potential through an artificial reprogramming process, which is also referred to as iPSC (induced pluripotent stem cells).

The term ' adult stem cells ' is a primitive cell just before differentiation, isolated from the tissues of mammals, including humans, and has the ability to grow indefinitely and various types of cells (eg, adipocytes, cartilage Cells, muscle cells, bone cells, etc.).

The ' extract ' referred to in the present invention refers to a substance extracted or separated from a raw material by an arbitrary method, and includes an extract obtained from a raw material, a concentrate obtained therefrom, a dried product and a powder of the concentrate, It means.

In addition, ' proliferation ' of stem cells referred to in the present invention is a concept distinct from the differentiation of stem cells, in which the stem cells are not differentiated into specific cells, and the cells are cleaved while maintaining the characteristics of the stem cells Which means that the total number of cells is increased.

Also, the term ' medium ' referred to in the present invention means a composition containing nutrients necessary for maintaining growth and survival of cells in vitro .

The term " subculture " referred to in the present invention means a method in which a part of cells is periodically transferred to a new culture container in order to continuously cultivate the cells in a healthy state for a long period of time, and the culture medium is continuously changed while the culture medium is changed . As the number of cells increases in a culture container having a limited space, it is used as a method for increasing the number of healthy cells, since proliferation nutrients are consumed or pollutants are accumulated and cells naturally die after a certain period of time. Usually, Culture vessel) or culturing the cell group separately is referred to as 1 passage.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention provides a composition for stimulating stem cell proliferation comprising an extract of Bleach as an active ingredient.

The white paper is a root of Angelica dahurica . In the present invention, the white paper is preferably used as a raw material of the composition for promoting stem cell proliferation, but the present invention is not limited thereto, and the Angelica genus, Plants of other species, which have high genetic affinity with Guritii as a plant, can also be used as a raw material for the active ingredient of the composition for promoting stem cell proliferation of the present invention. Plants having a high degree of flexibility in relation to the above-mentioned white papillae, such as coriander or perennial plant, may be cultivated or commercially available.

The stem cell proliferation promoting composition of the present invention contains the extract obtained from the above-mentioned white paper as an active ingredient. The extract can be obtained by any of known extraction methods such as a solvent extraction method, an ultrasonic extraction method, a filtration method and a reflux extraction method, and can be obtained by using a solvent extraction method or a reflux extraction method.

Water, an organic solvent, or a mixture thereof may be used as an extraction solvent for the extraction, and water is preferably used, but is not limited thereto. The organic solvent may be selected from the group consisting of C ₁ -C ₄ lower alcohols, hexane (n-hexane), ether, glycerol, propylene glycol, butylene glycol, ethyl acetate and methyl acetate, As the C ₁ -C ₄ lower alcohol, ethanol or methanol is preferably used, but not limited thereto.

The extraction can be carried out by adding the extraction solvent in an amount of 1 to 20 times the total weight of the white paper, preferably 2 to 10 times, more preferably 3 to 5 times, , But not limited to. When the addition amount of the extraction solvent is out of the above range, the extraction itself is not properly performed or the active ingredient can not be sufficiently secured even if it is extracted.

The extraction can be carried out in a temperature range of 20 to 200 ° C, preferably in a temperature range of 50 to 150 ° C, more preferably in a temperature range of 80 to 120 ° C, but is not limited thereto. Maintaining this temperature range may be effective in preventing decay, discoloration, and kinking.

The extraction can be performed for 0.5 to 20 hours, preferably for 1 to 15 hours, more preferably for 2 to 10 hours, but is not limited thereto. If the extraction time is out of the above range, the extraction itself may not be performed properly or the active ingredient may not be sufficiently secured even if it is extracted.

In addition, the extraction may be repeated several times, preferably one or two to five times, more preferably two to three times, but is not limited thereto.

After such an extraction process, the extract may be further roughened, such as concentrated or lyophilized. Specifically, the concentration may be concentrated under reduced pressure and concentrated using a vacuum rotary concentrator, but is not limited thereto. In addition, the concentration may be performed at 20 to 100 ° C, preferably 50 to 70 ° C, but is not limited thereto. In addition, the drying can be performed by lyophilization, and a vacuum freeze dryer is preferably used, but is not limited thereto. The lyophilization may be carried out in a temperature range of -50 to -100 ° C, preferably -60 to -80 ° C, but is not limited thereto.

The white bark extract obtained through the above process exhibits an effect of promoting the proliferation of stem cells. In other words, the blanched extract has an effect of accelerating the native proliferation rate of the stem cell itself more rapidly. In particular, the blanched extract is characterized in that stem cells are not differentiated and the number of stem cells is increased more rapidly while maintaining the characteristics of the stem cells.

The stem cell may be a stem cell derived from a mammal including a human. In addition, the stem cells may be adult stem cells, more preferably adult stem cells derived from gingivae.

In addition, the blanched extract of the present invention may be contained in an amount of 0.0001 to 120 μg / ml, preferably 0.0005 to 110 μg / ml, more preferably 0.001 To 100 μg / ml, but the present invention is not limited thereto. When the white fly extract is out of the above range, the stem cell proliferation promoting effect may be inhibited.

 In a specific example of the present invention, 2000 ml of water was added to 500 g of white paper, and the mixture was immersed for 2 hours and heated under reflux for 2.5 hours. The obtained white matter water extract was added to the gum-derived adult body at a concentration of 0.001 to 100 / / When the stem cells were treated with the stem cells, the stem cells were observed to promote their proliferation while maintaining their characteristics (see FIGS. 1 to 3) (see FIGS. 4 and 5).

Another aspect of the present invention provides a culture medium for stem cell culture comprising the above-mentioned blank white extract.

The blanched extract may be contained as one component in a culture medium for stem cell culture.

The culture medium for stem cell culture includes all mediums commonly used for culturing stem cells. For example, DMEM (Dulbeco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI1640, F Such as, but not limited to, -10, F-12, Minimal Essential Medium (MEM), Glasgow's Minimal Essential Medium (GMEM), and Iscove's Modified Dulbecco's Medium.

Another aspect of the present invention provides a method for promoting stem cell proliferation comprising the step of treating stem cells with the blanched extract.

The method of promoting stem cell proliferation of the present invention comprises the steps of treating the stem cell with the white skin extract and culturing the stem cell treated with the white skin extract.

The stem cells to be subjected to the proliferation promotion may be in vitro , and the white matter extract may be processed into stem cells in the form of a culture medium for culturing the stem cells.

The cultivation can be performed in an in-vitro state or an in-vivo state, and can be performed in a CO ₂ incubator, particularly when performed in an in-vitro state.

The culture in the CO ₂ incubator can be performed in an environment with a CO ₂ concentration of 5 to 15%, preferably 5 to 10%, more preferably 5 to 8% It is not limited. The inside of the CO ₂ incubator may be filled with O ₂ in addition to the CO ₂ .

In addition, the cultivation can be carried out in a temperature range of 25 to 45 DEG C, preferably 30 to 40 DEG C, more preferably 34 to 37 DEG C, but is not limited thereto. When the incubation temperature is outside the above temperature range, stem cell proliferation is not efficiently promoted and the number of stem cells to be killed is increased.

In addition, the culturing can be carried out continuously for 1 to 20 days, preferably 1 to 15 days, more preferably 1 to 10 days, but is not limited thereto. When the culture period is longer than 20 days, the nutrients are rapidly depleted and the characteristics of the cells are changed as the density of cells in the culture vessel is increased. In addition, subculture may be performed during the incubation period to separate the cell groups and cultivate them.

Hereinafter, the present invention will be described in detail with reference to Production Examples, Examples and Experimental Examples.

However, the following Production Examples, Examples and Experimental Examples are for illustrating the present invention, and the contents of the present invention are not limited by the following Production Examples, Examples and Experimental Examples.

[ Example  And Experimental Example ]

Preparation of Blank Paper Extract

2,000 ml of distilled water was added to 500 g of dried root of Angelica dahurica Bentham et Hooker and precipitated for 2 hours and then heated at 100 캜 under reflux for 2.5 hours to obtain a white water extract.

The extract was centrifuged at 5,000 x g for 10 minutes, and the supernatant was concentrated under reduced pressure to 300 ml using a rotary evaporator (Eyela NE-1001, Tokya Rikakikai Co. Ltd., Japan). The concentrate was lyophilized using a freeze dryer (Labconco, USA) to give 182.5 g of a solid residue (yield: 36.5% (w / w)).

Isolation and culture of gingival stem cells

Approved by the Ethical Review Committee of the Medical Research Ethics Committee of the Catholic University Medical College Seoul, we obtained normal gingiva tissue from four healthy patients undergoing crown enlargement.

The gingival tissues were immediately placed in sterile phosphate buffered saline (PBS, Welgene) containing 100 U / ml penicillin (Sigma Aldrich, USA) and 100 ug / ml streptomycin (Sigma Aldrich, USA) at 4 ° C. The gingival tissues were de-epithelialized and pulverized, digested with collagenase IV (Sigma Aldrich), and then incubated at 37 ° C in a humidified incubator of 5% CO ₂ and 95% O ₂ . After 24 hours of incubation, unattached cells were washed and replaced with medium based on? -MEM medium. The medium was prepared by mixing 15% fetal bovine serum (Gibco), 100 U / ml penicillin, 100 ug / ml streptomycin, 200 mM L-glutamine (Sigma Aldrich, USA) and 10 mM ascorbic acid 2- phosphate (Sigma Aldrich, ). Thereafter, human adult gingival adult stem cells were established by changing the medium every 2-3 days.

Determine whether stem cells are differentiated after treatment with blank white extract

Example 2 2.0 the stem cells established in 96 well plates × 10 ³ cells / arranged at a density of wells, the stem cells of Example 1 α-MEM medium containing the blank extracts obtained in (Gibco, USA ) In a humidified incubator of 5% CO ₂ and 95% O ₂ at 37 ° C. The α-MEM medium was prepared by mixing 15% fetal bovine serum (Gibco), 100 U / ml penicillin, 100 μg / ml streptomycin, 200 mM L-glutamine (Sigma Aldrich, USA) and 10 mM ascorbic acid 2- (Control), 0.001, 0.01, 0.1, 1, 10 and 100 μg / ml in the α-MEM medium. The morphology of the cells was confirmed on day 1, day 3 and day 7 using an inverted microscope (Leica DM IRM, Germany).

As a result, in the case of the stem cells cultured in the medium containing the blanched extract at a concentration of 0.001 to 100 μg / ml, the cells were spindle-shaped in the same manner as the control without the blank extract for 1 day to 7 days after the culture, (See Fig. 1 to Fig. 3).

From the above results, it can be seen that the differentiation does not progress in the stem cells during culturing with addition of the blanched extract of the present invention.

Confirming the effect of promoting the proliferation of stem cells after treatment with white-fly extract

Stem cells were cultured in the same manner as in Example 3, and living cells were identified using cell counting kit 8 (CCK.8, Japan) on days 1 and 7 of culture. Cell counting kit is an analytical method for confirming cell viability. It is based on the principle that water-soluble tetrazolium salt is reduced by dehydrogenase in cell in culture medium to become formazan, and the amount of formazan produced is living Since the amount of formazan produced is proportional to the number of cells, the degree of cell viability can be confirmed by confirming the amount of formazan by colorimetric assays. Cells at day 1 and day 7 of culture were separated from the culture vessel to prepare a cell suspension, and 100 μl per well of the cell suspension was added to a 96-well plate, and then 10 μl of CCK-8 solution was added to each well. Gt; 37 C < / RTI > for 2 hours in a humidified incubator of 5% CO ₂ and 95% O ₂ . The degree of proliferation of the stem cells was confirmed by measuring the spectrophotometric absorbance of the cultured 96-well plate using a microplate reader (BioTek, USA). The analysis was repeated three times.

As a result, when the stem cell density of the control group was set to 100% (100.0 ± 1.8) and the culture was carried out by adding 0.001, 0.01, 0.1, 1, 10 and 100 μg / Cell density was 102.5 ± 0.6, 133.3 ± 9.6%, 148.4 ± 20.5, 147.7 ± 12.6, 132.3 ± 27.7 and 101.1 ± 4.6%, respectively (see FIG. 4). In addition, when the stem cell density of the control group was 100% (100.0 ± 9.0) and the culture was performed by adding 0.001, 0.01, 0.1, 1, 10 and 100 μg / The density was 94.9 ± 22.3, 102.8 ± 22.1, 127.4 ± 7.4, 130.4 ± 1.3, 129.2 ± 10.8 and 124.8 ± 9.1%, respectively (see FIG. 5).

From the above results, it can be seen that stem cell proliferation is promoted by the treatment of the blanched extract, and in particular, when the blanched extract is contained in an amount of 0.1 or 1 μg / ml, have.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the scope of the invention is not limited to the disclosed exemplary embodiments. It will be possible to change it appropriately.

Claims (11)

A composition for stimulating proliferation of stem cells, comprising an extract of a blanched paper as an active ingredient. The method according to claim 1,
The above-mentioned blanched extract is extracted with water, an organic solvent or a mixture thereof as an extraction solvent. The method of claim 2,
Wherein the organic solvent is selected from the group consisting of C ₁ -C ₄ alcohols, hexane (n-hexane), ether, glycerol, propylene glycol, butylene glycol, ethyl acetate and methyl acetate. The method of claim 2,
Wherein the extraction solvent is water. The method according to claim 1,
Wherein the stem cell is an adult stem cell. The method of claim 5,
Wherein said adult stem cells are derived from gingiva. The method according to claim 1,
Wherein the blanched extract is contained in an amount of 0.0001 to 120 占 퐂 / ml. A culture medium for stem cell culture comprising a blank white extract. And treating the stem cells with a blank white extract. The method of claim 9,
Wherein the white paper extract is extracted with water, an organic solvent or a mixture thereof as an extraction solvent. The method of claim 10,
Wherein the extraction solvent is water.

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