KR20190112559A - Composition for Promoting the Differentiation of Stem Cells Comprising the Extract of Pongam tree - Google Patents

Abstract

The present invention relates to a composition for promoting stem cell differentiation using an active ingredient derived from pongam tree, and a method for promoting differentiation of stem cells using the composition. The pongam tree extract has an effect of inducing bone differentiation, and thus can be usefully used for bone differentiation of stem cells for the purpose of cell therapy.

Description

Composition for Promoting the Differentiation of Stem Cells Comprising the Extract of Pongam tree}

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

Recently, the use of stem cells in regenerative medicine using tissue engineering has been proposed as a new field for the treatment of intractable disease. Accordingly, interest in stem cell research has increased, and it is recognized that the use of stem cells capable of forming tissues through proliferation and differentiation can solve problems caused by tissue damage as well as treatment of various diseases.

Stem cells have the ability to self-replicate in undifferentiated state and have the property of differentiating to specific cells under appropriate conditions. Stem cells can be divided into embryonic stem cells and adult stem cells, depending on their origin. Since human embryonic stem cells are obtained from embryos that can occur as human organisms, they have excellent cell proliferation and differentiation, but have bioethical problems. In contrast, adult stem cells have limited differentiation capacity than embryonic stem cells, but ethical problems are reduced because cells that are already present in various organs of the human body are collected from bone marrow, blood, brain, and skin and developed into stem cells. . Adult stem cells include hematopoietic stem cells, mesenchymal stem cells, neuroblastoma cells, epithelial hair cells, etc., and hematopoietic stem cells are capable of producing blood cells and lymphocytes, and are useful for treating immune system diseases including blood cancer. Stem cells differentiate into bone, cartilage, adipose and fibrous tissues and are involved in their recovery when each tissue is damaged. However, these adult stem cells are present in small amounts in their tissues and may not exist or divide for many years.

In other words, the stem cells refers to 'undifferentiated' cells that have such differentiation ability but have not yet differentiated. In this undifferentiated state, if appropriate conditions are met, stem cells can be differentiated into various tissue cells, and researches on the treatment of diseases using them are being actively conducted, but as ethical problems of embryonic stem cells emerge, relatively ethical Attempts to use adult stem cells with fewer problems have attracted attention.

Therefore, human stem cells are separated from various tissues including bone marrow, periosteum, adipose tissue and muscle. Previous studies have shown that mesenchymal stem cells derived from gingival tissue may be a new source for stem cell-based therapies in bone regeneration, such as clinically bone reconstruction. In addition, human gingival tissue-derived mesenchymal stem cells have been shown to be superior to bone marrow-derived mesenchymal stem cells for regenerative medicine in regenerative medicine (Tomar GB, Srivastava RK, Gupta N, Barhanpurkar AP, Pote ST, Jhaveri HM, et al. Human gingiva-derived mesenchymal stem cells are superior to bone marrow-derived mesenchymal stem cells for cell therapy in regenerative medicine.Biochem Biophys Res Commun 2010; 393: 377-383.). Like other sources of stem cells, immunomodulatory properties have been found in gingival tissue-derived mesenchymal stem cells. Stem cells derived from gingival tissue have several advantages, including ease of isolation, accessible tissue sources, and rapid ex vivo expansion.

On the other hand, certain herbal extracts that have long been used in traditional Asian medicine have been shown to be a source of new therapeutics (Jeong SH, Lee JE, Jin SH, Ko Y and Park JB: Effects of Asiasari radix on the morphology and viability of mesenchymal stem cells derived from the gingiva.Mol Med Rep 10: 3315-3319, 2014.). Such extracts can be considered pre-validated to validate and are expected to have fewer safety problems than chemical synthetic drugs. Therefore, the researchers of the present invention devised the present invention to increase the viability and differentiation of stem cells, which is safer and more effective than chemical synthetic drugs.

Chinese Patent Publication CN103330733A

An object of the present invention is to provide a composition for promoting stem cell differentiation using an active ingredient derived from Pongam tree, and a method for promoting differentiation of stem cells using the composition.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task not mentioned will be clearly understood by those skilled in the art from the following description.

In order to solve the above problems, the present invention provides a composition for promoting stem cell differentiation and a culture medium for stem cell comprising a Pongam tree extract as an active ingredient.

In another aspect, the present invention provides a method for promoting stem cell differentiation comprising the step of treating Pongam tree extract to stem cells.

The stem cell differentiation promoting composition provided by the present invention uses a Pongam tree extract derived from natural products as an active ingredient, thereby reducing cytotoxicity and side effects due to chemicals, and effectively differentiating stem cells to specific cells. Because it can be enhanced, it is expected to be widely used as a composition for differentiating various stem cells into specific cells.

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

1 is a photograph showing the morphological changes of cells on the day 1 after culture after treatment with adult stem cells derived from gingham (Pongam) tree according to an embodiment of the present invention (initial magnification: 200 times, scale bar : 200 mu m);
A: control group, B: 0.001% by weight, C: 0.01% by weight, D: 0.1% by weight, E: 1% by weight.
Figure 2 is a graph showing the cell viability of day 1 culture after Pongam tree extract treatment according to an embodiment of the present invention.
Figure 3 is a graph showing the degree of ALP activity of the 3 and 7 days of culture after treatment with Pongam tree extract according to an embodiment of the present invention.
FIG. 4 is a photograph (initial magnification: 100 times, scale bar: 400 μm) of observing the degree of mineralization of cultured 3 days after Pongam tree extract treatment according to an embodiment of the present invention.
Figure 5 is a photograph (initial magnification: 100 times, scale bar: 400㎛) observed the degree of mineralization of 7 days of culture after treatment with Pongam tree extract according to an embodiment of the present invention.
Figure 6 is a graph showing the degree of mineralization of the 3 and 7 days of culture after treatment with Pongam tree extract according to an embodiment of the present invention.

Hereinafter, various embodiments described herein are described with reference to the drawings. In the following description, for a thorough understanding of the present invention, various specific details are set forth, such as specific forms, compositions, processes and the like. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and forms. In other instances, well known processes and manufacturing techniques have not been described in particular detail in order to not unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "embodiment" means that a particular feature, form, composition or characteristic described in connection with the embodiment is included in one or more embodiments of the invention. Thus, the context of "in one embodiment" or "embodiment", represented at various places throughout this specification, does not necessarily represent the same embodiment of the invention. In addition, particular features, forms, compositions, or properties may be combined in any suitable manner in one or more embodiments.

Unless otherwise defined in the present invention, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In the present invention, the "stem cell" is a cell having the ability to differentiate into two or more cells while having a self-replicating capacity, and according to differentiation capacity, totipotent stem cells, pluripotent stem cells (pluripotent) stem cells) and multipotent stem cells.

"Gingiva" according to the present invention may be a more advantageous source of stem cells. Stem cells have self-renewal and pluripotency with the ability to differentiate into various cell types, including bone, adipose tissue and cartilage. Stem cells can be obtained from a variety of sources, including oral regions (Ballini A, De Frenza G, Cantore S, et al: In vitro stem cell cultures from human dental pulp and periodontal ligament: new prospects in dentistry.Int J Immunopathol Pharmacol 20: 9-16, 2007). Dental pulp and periodontal ligament are considered preferred sources. However, due to the limited amount of tissue, additional tooth extraction procedures are required. Therefore, the researchers of the present application have stem cell markers including CD44, CD73, CD90, and CD105 that gingival stem cells possess bone formation ability, plastic adhesion and multiple line differentiation ability to form bone formation, fat formation and cartilage formation. It was found to express. In addition, harvesting stem cells from the gingiva has the advantage of being easier than the bone marrow of the maxilla and mandible due to invasiveness, pain and loss of sensation.

In addition, in the present invention, "extract" refers to a material extracted or separated from the raw material by any method, and means including all of the extract extracted from the raw material, the concentrate obtained therefrom, the dried product and the powder of the concentrate without limitation. to be. Several methods can be applied to herbal extraction. For example, extraction methods, such as cold extrusion, compression pressing, and solvent extraction, can be used, but are not limited to these. Various solvents can be used for solvent extraction such as ethanol, methanol, butanol, hexane, methylene chloride, ethyl acetate and sterile water (Wang MH, Jeong SH, Guo H and Park JB: Anti-inflammatory and cytotoxic effects of methanol, ethanol , and water extracts of Angelicae Dahuricae Radix.J Oral Sci 58: 125-131, 2016). A new microwave assisted extraction method for Pongamia pinnata extraction has also been developed (Soumya V, Muzib YI and Venkatesh P: A novel method of extraction of bamboo seed oil (Bambusa bambos Druce) and its promising effect on metabolic symptoms of experimentally induced polycystic ovarian disease.Indian J Pharmacol 48: 162-167, 2016). Different solubilities and different boiling points appear between different types of solvents. It should be noted that different effects may be achieved depending on the type and concentration of solvent.

As used herein, the term "differentiation" refers to a phenomenon in which cells divide and proliferate, and the structure or function of the cell is specialized while the entire individual is growing. In other words, it refers to a process in which cells, tissues, etc. of an organism are transformed into suitable forms and functions to play a role given to each. For example, the process of transforming pluripotent stem cells such as embryonic stem cells into ectoderm, mesoderm, and endoderm cells is also differentiation, and in particular, the process of turning hematopoietic stem cells into erythrocytes, leukocytes, platelets, etc. also corresponds to differentiation. The present invention devised a composition containing a novel active ingredient that promotes differentiation using stem cells derived from human gingival tissue, and also found a concentration having excellent effects.

In addition, in the present invention, "medium" means a composition containing a nutrient required to maintain the growth and survival of cells in vitro, and generally known media such as RPMI-1640, DMEM, MEM, Or some component thereof, and may also include glutamine, fetal calf serum, and the like, but is not limited thereto if the cell includes components necessary for maintaining growth and survival.

In the present invention, "passage culture" refers to a method of continually cultivating the generation of cells while periodically transferring a portion of the cells to a new culture vessel in order to continuously cultivate the cells in a healthy state for a long time. it means. It is used as a method to increase the number of healthy cells, since the number of cells increases in a culture vessel with a limited space, and proliferation nutrients are consumed or contaminants accumulate, causing the cells to die naturally. Replacing the (culture vessel) or dividing the cell population into cultures is called one passage.

One aspect of the present invention relates to a stem cell differentiation promoting composition comprising a Pongam tree extract as an active ingredient.

The "Pongam tree" is generated in India as a raw material of the active ingredient of the composition for increasing stem cell viability and promoting differentiation in the present invention. Pongam trees are also called Indian beech trees or Pongam oil trees. In general, Pongam trees are widely used to treat rheumatism, and are extracted from brown oil seeds.

In one embodiment of the present invention, the Pongam tree may be Pongamia pinnata.

The Pongamia pinnata is a soybean (Fabaceae native to tropical and temperate Asia, including parts of India, China, Japan, Malaysia, Australia and the Pacific Island as a raw material of the composition for increasing stem cell viability and promoting differentiation in the present invention. It is a tree belonging to. It is also recently moved into Millettia and is also used synonymously with Millettia pinnata. M. pinnata is primarily used as a natural pesticide.

In the present invention, it is preferable to use the Pongamia pinnata as a raw material of the active ingredient of the composition for promoting stem cell differentiation, but not limited thereto, and other species of plants of high genetic flexibility with the Pongamia pinnata may also be derived from the stem cell differentiation of the present invention. It can be used as a raw material of the active ingredient of the composition for promotion.

Stem cell differentiation promoting composition of the present invention comprises a Pongam (Pongam) tree extract as an active ingredient.

Pongam tree used in the present invention for the production of the extract is not limited to the entire range of the plant, such as its roots, berries, stems or leaves, and the Pongam tree is cultivated herein Or a commercially available one.

The extract may be obtained through all conventionally known extraction methods such as solvent extraction, ultrasonic extraction, filtration and reflux extraction, and may be preferably obtained using solvent extraction or reflux extraction.

In the extraction, water, an organic solvent or a mixture thereof may be used as the extraction solvent, preferably alcohol, more preferably methanol, but is not limited thereto. The organic solvent may be selected from the group consisting of C1-C4 lower alcohol, hexane (n-hexane), ether, glycerol, propylene glycol, butylene glycol, ethyl acetate and methyl acetate, but is not limited thereto. As the lower alcohol of C4, ethanol or methanol is preferably used, but is not limited thereto.

The extraction may be carried out by adding the extraction solvent 1 to 30 times the total weight of the Pongam tree, preferably 3 to 20 times, more preferably 5 to 10 times It may be, but is not limited thereto. When the amount of the extraction solvent added is out of the range, there is a problem that the active ingredient is not sufficiently secured even if the extraction itself is not properly performed or extracted.

The extraction may be carried out at room temperature or warming conditions, preferably at about 20 to 200 ℃, more preferably at a temperature of about 50 to 150 ℃, but is not limited thereto. The extraction time is preferably about 1 to 15 hours, more preferably about 2 to 10 hours, but is not limited thereto, and may vary depending on conditions such as extraction solvent and extraction temperature. The extraction may be extracted one or more times several times in order to obtain a larger amount of the active ingredient, preferably one to five times, more preferably three times the continuous extraction can be used combined extract.

After the extraction process as described above, the extract may additionally undergo a step such as concentration or lyophilization. Specifically, the concentration may be concentrated under reduced pressure, preferably concentrated using a vacuum rotary concentrator, but is not limited thereto. In addition, the concentration may be carried out at 20 ~ 100 ℃, preferably at 50 ~ 70 ℃, but is not limited thereto. In addition, the drying may be lyophilized, preferably using a vacuum freeze dryer, but is not limited thereto. In addition, the lyophilization may be carried out in a temperature range of -50 ~ -100 ℃, preferably may be carried out in a temperature range of -60 ~ -80 ℃, but is not limited thereto.

In one embodiment of the present invention, the extraction solvent used methanol. As extraction method, it can extract by various methods, such as cold extrusion, compression extraction, and solvent extraction. Solvent extraction may use a variety of solvents such as ethanol, methanol, butanol, hexane, methylene chloride, ethyl acetate and sterile water, but is not limited thereto. In addition, when applying the compression extraction method, it is preferable to apply a relatively high temperature as compared to cold extrusion.

Pongam tree extract obtained through the above process has the effect of promoting the differentiation of stem cells into osteoblasts. In other words, the Pongam tree extract induces stem cells to differentiate into osteoblasts, and differentiated osteoblasts exhibit high alkaline phosphatase (ALP) activity and form mineral crystals.

The stem cells may be stem cells derived from mammals, including humans. In addition, the stem cells may be adult stem cells, more preferably may be adult stem cells derived from gingiva.

In addition, the stem cell differentiation promoting composition of the present invention may include the Pongam tree extract in an amount of 0.0005 to 0.9% by weight, preferably in an amount of 0.001 to 0.5% by weight, more preferably May be included in an amount of 0.001 to 0.1% by weight, but is not limited thereto if it has a concentration capable of differentiating stem cells.

Another aspect of the invention relates to a stem cell culture medium comprising a Pongam tree extract as an active ingredient.

The Pongam tree extract may be included as one component in a culture medium for stem cell culture.

The stem cell culture medium includes all mediums conventionally used for culturing stem cells, and for example, DMEM (Dulbeco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI1640, F -10, F-12, basic essential medium (MEM), Glasgow's Minimal essential Medium (GMEM), Iscove's Modified Dulbecco's Medium (IMDM), or a medium containing components that can induce bone differentiation, This is not limitative.

Another aspect of the invention relates to a method for promoting stem cell differentiation comprising the step of treating a Pongam tree extract to stem cells.

The method of promoting stem cell differentiation of the present invention includes treating the Pongam tree extract to stem cells and culturing the stem cells treated with the Pongam tree extract.

Stem cells that are the target of promoting bone differentiation may be in vitro, and the Pongam tree extract may be treated to stem cells in the form of the stem cell culture medium. In addition, stem cells may be cultured in a three-dimensional support to promote bone differentiation. The components constituting the support may use a biocompatible material, and osteoblasts cultured and differentiated from the support may be implanted in a living body attached to the support for the treatment of bone diseases without separating from the support.

The culturing can be carried out in vitro or in vivo, and especially in the CO ₂ incubator, if performed in vitro.

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

In addition, the culturing may be carried out in a temperature range of 25 to 45 ℃, preferably may be carried out in a temperature range of 30 to 40 ℃, more preferably 34 to 37 ℃, but is not limited thereto. When the culture temperature is out of the above temperature range, the differentiation of stem cells is not promoted efficiently, and there is a problem that many stem cells are killed.

In addition, the culture may be performed continuously for 1 to 25 days, preferably 1 to 15 days, more preferably 1 to 10 days, but is not limited thereto. When the culture period exceeds 25 days, as the density of cells in the culture vessel increases, the nutrient component is quickly depleted, the characteristics of the cells change, and differentiation into osteoblasts is not induced. In addition, subcultures may be performed during the incubation period to divide the cell groups.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example 1 Preparation of Pongam Tree Extract

Pongamia pinnata (L.) Pierre used a sample from Bangladesh, and the specimen was deposited at the Korea Biotechnology Institute's Botanical Repository (Accession No. PB022015). The powder of 58 g of dried and pulverized Pongamia pinnata (PPT) leaves was added to 500 ml of methanol, and extracted by refiltration at room temperature.

This methanol extract was filtered at room temperature and concentrated to 60 ml using a rotary distillation machine (Eyela NE-1001, Tokya Rikakikai Co Ltd, Japan) under reduced pressure. The concentrate was lyophilized using a lyophilizer (Labconco, USA) to give 15.04 g of solid residue (yield: 26% (w / w)).

Example 2 Isolation and Culture of Gingi-Derived Stem Cells

After approval by the Medical Research Ethics Review Board of the Catholic University of Korea, Seoul St. Mary's Hospital (no. KC11SISI0348), normal gingiva tissue was obtained from four healthy patients undergoing angioplasty.

The gingival tissue was immediately placed in sterile phosphate buffered saline (PBS, Welgene) containing 100 U / ml penicillin (Sigma Aldrich, USA) and 100 μg / ml Streptomycin (Sigma Aldrich, USA) and placed at 4 ° C. The gingival tissue was de-epithelialized and cut into 1-2 mm, 1 mg / ml of dispase (Sigma Aldrich, USA) and 2 mg / ml collagenase IV (Sigma Aldrich, USA) After digestion with, it was incubated at 37 ° C. in a wet incubator with 5% CO ₂ and 95% O ₂ . After 24 hours of culture, unattached cells were washed away and replaced with a medium based on α-MEM medium. The medium contained 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-phosphate (Sigma Aldrich, USA). ). Subsequently, adult stem cells derived from human gingiva were established by changing the medium every 2-3 days.

Example 3 After the Pongam Tree Extract Treatment, Confirm the Stem Cell Morphology

The stem cells established in Example 2 were placed in a 96-well plate at a density of 2.0 × 10 ³ cells / well, and the stem cells were added to α-MEM medium to which the Pongam tree extract obtained in Example 1 was added ( Gibco, USA) was incubated at a temperature of 37 ° C. in a wet incubator of 5% CO ₂ and 95% O ₂ . The α-MEM medium contained 15% fetal bovine serum (Gibco), 38 ug / ml dexamethasone, 2 mg / ml glycerophosphate disodium hydrochloride, 100 U / ml penicillin and 100 µg / ml streptomycin (Sigma Aldrich, USA), 200 mM L-glutamine (Sigma Aldrich, USA) and 10 mM ascorbic acid 2-phosphate (Sigma Aldrich, USA), wherein the Pongam tree extract is dissolved in 0.1% dimethyl sulfoxide (DMSO) After the final concentration of 0 (control), 0.001%, 0.01%, 0.1% and 1% by weight in the α-MEM medium was added. The morphology of the cells was confirmed by inverted microscope (CKX41SF; Olympus Co., Ltd., Japan) on day 1 to obtain an image.

As a result, in the case of stem cells cultured in a medium containing 0.001 to 1% of Pongam tree extract, the cell type was the same spindle type as the control group without adding the Pongam tree extract on day 1 after the culture. shaped), it was confirmed that the fibroblast-like morphology, the Pongam tree extract of the present invention has no toxicity affecting the stem cells, the adhesion is not reduced (Fig. 1).

Example 4 After the Pongam Tree Extract Treatment, Stem Cell Survival Confirmation

Stem cells were cultured in the same manner as in Example 3, and the living cells were identified using the cell count kit-8 (CCK8, Dojindo, Japan) on the day of culture. The cell counting kit is an analytical method for checking cell viability, using the principle that the water-soluble tetrazolium salt is reduced by dehydrogenase of cells in the culture medium to form formazan, and the amount of formazan produced is alive. Since it is proportional to the number of cells, the viability of the cells can be confirmed by confirming the production amount of the formazan by colorimetric assays. The cells of the day 1 of culture were separated from the culture vessel to make a cell suspension, the cell suspension was put in a 100 well per well in a 96 well plate, and then 10 μl of CCK-8 solution was added to each well. Incubated at 37 ° C. for 2 hours in a wet incubator with 5% CO ₂ and 95% O ₂ . The 96-well plate in which the culture was completed was measured by spectroscopic optical absorbance at 450 nm using a microplate reader (BioTek, USA) to confirm the degree of survival of stem cells.

As a result, when the stem cell density of the control group was 100 wt% (100.0 ± 5.1%) (P> 0.05) on day 1, the Pongam tree extract was 0.001 wt%, 0.01 wt%, 0.1 wt%, and The group treated with 1% by weight showed 94.0 ± 5.4, 94.3 ± 7.2, 91.1 ± 4.9, and 86.5 ± 5.2, confirming that the Pongam tree extract of the present invention had no toxicity affecting stem cells. (Figure 2)

Example 5 Alkaline phosphatase (ALP) Activity Level Confirmation after Pongam Tree Extract Treatment

ALP is known as a marker of osteoblast activity and acts as a regulator of inorganic phosphate transport, cell division or differentiation during calcification. Therefore, ALP activity was measured to determine whether Pongam tree extract induces the differentiation of gingival adult stem cells into osteoblasts.

Stem cells were cultured in the same manner as in Example 3, and replaced with fresh medium every 3-4 days. ALP activity was measured according to the manufacturer's recommended method using commercially available kits (K412-500, BioVision, Inc., Milpitas, Calif., USA) on days 3 and 7 of culture and using a microplate reader (BioTek, USA) The absorbance change by p-nitrophenyl phosphate at 405 nm was used as an index.

As a result, the absorbance of the group treated with 0% by weight (control), 0.001%, 0.01%, 0.1% and 1% by weight of Pongam tree extract was 0.073 ± 0.000, 0.075 ± 0.001 on the 3rd day of culture. , 0.083 ± 0.000, 0.078 ± 0.002, and 0.081 ± 0.001, and 0.104 ± 0.001, 0.110 ± 0.000, 0.122 ± 0.001, 0.130 ± 0.001 and 0.121 ± 0.000 on the 7th day of culture (Table 1). ) The treatment of the tree extract significantly increased the ALP activity compared to the control group (FIG. 3). In particular, when the concentration of Pongam tree extract was 0.1% by weight, it showed the highest activity.

Pongam Tree Extract 0% by weight
(Control) 0.001% by weight 0.01 wt% 0.1 wt% 1 wt% 3-day absorbance 0.073 0.075 0.083 0.078 0.081 7-day absorbance 0.104 0.11 0.122 0.130 0.121 Increase 42.466% 46.667% 46.988% 66.667% 49.383%

As shown in Table 1 above, using the percentage of the ratio of the three-day absorbance to the seven-day absorbance, the increase rate of ALP activity according to the treatment concentration of Pongam tree extract was confirmed, compared with the control, It was found to increase at 0.001%, 0.01%, 0.1% and 1% by weight.

Example 6: Confirm the degree of mineralization after Pongam tree extract treatment

Alizarin red binds to mineral crystals such as calcium. Since osteoblasts form mineral crystals, the degree of differentiation into osteoblasts can be known in proportion to the degree of alizarin red staining. The present inventors performed alizarin red staining after culturing the stem cells in a medium containing the Pongam tree extract in order to determine whether the stem cells are differentiated into osteoblasts by the Pongam tree extract.

Stem cells were cultured in the same manner as in Example 3, and replaced with fresh medium every 3-4 days. Alizarin Red S staining (ScienCell Research Laboratories, Inc., Carlsbad, CA, USA) was performed to measure calcium formation on days 3 and 7 of culture.

First, Alizarin Red S staining solution was prepared. 2 g of alizarin red S was dissolved in 100 ml of distilled water, and the pH was adjusted to 4.1 to 4.3 to prepare an alizarin red S staining solution, which was then filtered and stored in a dark place.

To fix the cultured cells, the culture medium was removed from the culture vessel and the cells were washed with PBS. After removal of PBS, neutralized formalin (10%) was treated to cover the cells. After 30 minutes, formalin was removed and the cells were washed with distilled water, followed by distilled water. Alizarin Red S prepared and stored above was added to the fixed cells and stained in the dark for 45 minutes. After completion of dyeing, alizarin red S was removed, washed four times with distilled water, and then phosphate buffer was added. Stained cells were observed with an inverted microscope (Olympus Corporation, CKX41SF) (Figure 4, Figure 5). In addition, in order to quantitatively evaluate the degree of calcium formation, Alizarin Red S bound to calcium was dissolved in 10 mM sodium phosphate containing 10% cetylpyridinium chloride (Sigma Aldrich, USA), followed by a spectrophotometer (BioTek, USA). Was measured.

As a result, the absorbance of the group treated with 0% by weight (control), 0.001%, 0.01%, 0.1% and 1% by weight of Pongam tree extract was 0.059 ± 0.002, 0.044 ± 0.002 , 0.052 ± 0.005, 0.049 ± 0.004 and 0.063 ± 0.002, and 0.082 ± 0.002, 0.102 ± 0.001, 0.085 ± 0.002, 0.084 ± 0.007 and 0.080 ± 0.001 on the 7th day of culture (Table 2, Fig. 6). It was confirmed that the significant increase in absorbance was the highest in the 0.001% by weight group on the 7th day (P <0.05).

Pongam Tree Extract 0% by weight
(Control) 0.001% by weight 0.01 wt% 0.1 wt% 1 wt% 3-day absorbance 0.059 0.044 0.052 0.049 0.063 7-day absorbance 0.082 0.102 0.085 0.084 0.080 Increase 38.983% 131.818% 63.462% 71.429% 26.984

As shown in Table 2 above, using the percentage of the three-day absorbance to the 7-day absorbance, the increase in mineralization according to the treatment concentration of Pongam tree extract was confirmed, compared to the control, 0.001 weight It was found to increase in%, 0.01% by weight and 0.1% by weight.

The present invention provides for the effect of different concentrations of Pongam tree extract on osteogenic differentiation of stem cells derived from human gingival tissue. The present invention clearly demonstrates that short term application of Pongam tree extract enhanced the proliferation of mesenchymal stem cells.

By applying low dose Pongam tree extract, ALP activity was improved by 25% and mineralization by 24%. Different doses may have different effects on stem cells. ALP activity showed the highest activity in 0.1% by weight of Pongam tree extract, and it was confirmed that a significant increase in mineralization was achieved in 0.001% by weight of Pongam tree extract. Previous studies have shown that lower concentrations of traditional herbs increase total metabolic and ALP activity expression but significantly reduce cell populations at higher concentrations (Liu YR, Qu SX, Maitz MF, Tan R, Weng J). The effect of the major components of Salvia Miltiorrhiza Bunge on bone marrow cells.Journal of ethnopharmacology 2007; 111: 573-583). However, the present inventors found that the dose of Pongam tree extract may be different depending on the stage, system, and incubation period of the cultured cells in order to obtain the maximum effect.

It was confirmed that the Pongam tree extract according to the present invention can effectively promote bone formation differentiation of mesenchymal stem cells, and through this, the composition for promoting stem cell differentiation comprising the Pongam tree extract of the present invention. And it can be confirmed that it can be used in the method for promoting the differentiation of various stem cells using this.

Claims (16)

Pongam (Pongam) tree extract comprising as an active ingredient, the composition for promoting stem cell differentiation. The method of claim 1,
The Pongam tree is Pongamia pinnata, stem cell differentiation promoting composition. The method of claim 1,
The Pongam tree extract is extracted using water, an organic solvent or a mixture thereof as an extractant, the composition for promoting stem cell differentiation. The method of claim 3, wherein
The organic solvent is one or more organic solvents selected from the group consisting of C1-C4 alcohol, hexane (n-hexane), ether, glycerol, propylene glycol, butylene glycol, ethyl acetate and methyl acetate, for promoting stem cell differentiation Composition. The method of claim 4, wherein
The alcohol is methanol, a composition for promoting stem cell differentiation. The method of claim 1,
The stem cells are adult stem cells, composition for promoting stem cell differentiation. The method of claim 6,
The adult stem cells are derived from gingiva tissue, composition for promoting stem cell differentiation. The method of claim 1,
The composition comprises a Pongam tree extract in an amount of 0.0005 to 0.9% by weight, the composition for promoting stem cell differentiation. Pongam (Pongam) tree extract containing as an active ingredient, the culture medium for stem cells. The method of claim 9,
The Pongam tree is Pongamia pinnata, stem cell culture medium. The method of claim 9,
The stem cells are adult stem cells, stem cell culture medium. The method of claim 11,
The adult stem cells are derived from gingiva tissue, stem cell culture medium. The method of claim 9,
The medium comprises a Pongam tree extract in an amount of 0.0005 to 0.9% by weight, the culture medium for stem cells. A method for promoting stem cell differentiation, comprising the step of treating Pongam tree extract to stem cells. The method of claim 14,
The Pongam tree is Pongamia pinnata, a method for promoting stem cell differentiation. The method of claim 14,
The treating the Pongam tree extract is to treat the Pongam tree extract at a concentration of 0.0005 to 0.9% by weight, a method for promoting stem cell differentiation.

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