KR101777457B1 - The composition for improving the growth of human skin cells containing Juniperus chinensis extract as an active ingredient - Google Patents
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Abstract
The composition comprising the extract of the present invention as an active ingredient has the effect of improving the proliferation of human skin cells.
Description
The present invention relates to a composition for improving the proliferation of human skin cells, which comprises a jellyfish extract as an active ingredient.
The present invention also relates to a method for improving the proliferation of human skin cells, which comprises treating the isolated human skin cells with a bamboo extract.
The japanese tree is an evergreen needles belonging to the Quercus mongolica family. It reaches 15 to 25 meters tall and is a tree that smells from the whole body. The young japanese tree grows in a cone shape, and its stem is straight, but with its age it has the characteristic of being twisted and bent according to the surrounding conditions. Components of juniper trees include amentoflavone, hinokiflavone, and apigenin, and essential oils such as cedrol, pinene and the like are present. It was used as a fragrance because of its clear and strong fragrance. It was also used as a precious material to make various folk crafts such as chests, curtains, and so on.
On the other hand, the juniper has been used as a medicine for accelerating blood circulation, sterilizing action, detoxifying action, antipyretic action, cold, constipation and various diseases from the past, and researches for the treatment of various diseases . In recent years, research results have been reported that extracts of jellyfish are effective for protection of skin cells.
In one study, when skin cells were damaged by ultraviolet light or the like, the extract of mungbean extracts showed 98% or more free radical scavenging activity at a concentration of 100 μg / ml and inhibited MMP activity by 95% or more. In addition, the effect of hyaluronidase inhibition by 81% was observed at a concentration of 2 mg / ml, and it was reported that the fragrance extract of chestnut tree protects skin cells damaged by ultraviolet rays (J. Soc. Cosmet. 30 (1), 63-71 (2004)). Another study confirmed the antimicrobial efficacy of the juniper extract against four microorganisms that cause skin inflammation. As a result, it has been reported that when the concentration of the juniper extract is 250 μg / ml or more, the antimicrobial effect against the four microorganisms is confirmed, and the extract of the juniper has the effect of protecting human skin cells. 8 (2), 107-116 (2010)). However, even in the case of extracts of natural products, there is a possibility that the cytotoxicity may be exhibited at a high concentration of 100 μg / ml or more. In fact, when the concentration of the extract of the mungbean extract is 50 μg / ml or more, It was found to be abruptly decreased.
On the other hand, studies confirming the effect of protecting the skin cells against the extracts of juniper extracts have been reported regularly, but there have been no studies confirming the effect of proliferating skin cells, which are different from the effects of treating and protecting damaged skin.
Accordingly, the inventors of the present invention have studied natural extracts showing an effect of improving the proliferation of human skin cells. As a result, they have found that the extract of jellyfish enhances the proliferative capacity of human skin cells. In particular, in studies on the extract of juneaceae which protects existing skin cells, the present inventors have found that the low concentration of juneae extract is a safe substance free from cytotoxicity, and at the same time, it enhances the proliferation of human skin cells And the present invention has been completed.
The main object of the present invention is to provide a composition for improving the proliferation of human skin cells, which comprises a fragrant extract of cherry as an active ingredient.
Another object of the present invention is to provide a method for improving the proliferation of human skin cells, which comprises treating the isolated human skin cells with a jelly extract.
In one aspect of the present invention, there is provided a composition for improving the proliferation of human skin cells comprising an extract of Juniperus chinensis as an active ingredient.
The term in the invention "juniper (Juniperus chinensis ) "is an evergreen tree of the Japanese cabbage tree, and its branches and leaves are mainly used as medicines and have been reported to be effective for cold, arthritis, pain caused by wind and moisture.
In the present invention, the term "extract" means a product obtained by immersing a desired substance in various solvents and then extracting the liquid component obtained by extracting the product at room temperature or a constant temperature for a predetermined time, the solid component obtained by removing the solvent from the liquid component, It can mean. In addition, in addition to the above-mentioned results, the present invention can be broadly interpreted to include all of the diluted solution, the concentrated solution thereof, the adjusted product thereof, and the purified product. The method for obtaining the above extract is not particularly limited as long as it can obtain an extract having an effect of improving the proliferation of skin cells. Preferably, the roots, stems, leaves, dried products, A method such as a cold extraction method for extracting at a room temperature of 10 to 25, a heating extraction method for heating by 40 to 100, an ultrasonic extraction method for extracting by using ultrasonic waves, a reflux extraction method using a reflux condenser, etc. can be used.
In one embodiment of the present invention, the fragrant jelly bark extract was extracted with ethanol after pulverizing 5 kg of juniper seeds into powder. Specifically, five times the ethanol solvent of the juniper powder was added to the juniper powder, and the resultant was repeatedly extracted three times at 65 占 폚. Thereafter, the extracted sample was filtered, concentrated with a vacuum concentrator, and lyophilized to obtain a jellyfish extract.
In the present invention, the jukebox extract may have a concentration of 1 / / ml to 7 / / ml, and the concentration may be 1 / / ml to 5 / / ml. In addition, the concentration of the fragrance extract may be 5 μg / ml.
In one embodiment of the present invention, after culturing the cultured human skin cells, the juniper extract is added to the cultured cells at a concentration of 1, 5, 10, 25, 50, 75, 100, 150, Cells. Thereafter, when the survival rate of the cells was measured in combination with trypan blue exclusion method, it was confirmed that the extract was safe for cells without cytotoxicity when the concentration of juniper extract was 1 to 5 / / ml.
In addition, in an embodiment of the present invention, human skin cells were treated with 1, and 5 / / ml of juniper extract without cytotoxicity to evaluate cell proliferation ability. As a result, compared with the high concentration extract of 10 / / It was confirmed that the proliferation ability of human skin cells was improved. In particular, when the extract was treated with 5 / / ml of the juniper extract, the human skin cells showed the most excellent proliferative activity. On the other hand, in contrast to the above results, it was confirmed that in the group treated with 10 / / ml of juniper extract, the human skin cell proliferative capacity was remarkably decreased as compared with the group treated with 5 / / ml juniper extract.
In the present invention, the juniper extract may be treated with human skin cells for 1 to 3 days, but not limited thereto. Specifically, the juniper extract may be treated with human skin cells for 2 days.
In one embodiment of the present invention, human skin cells were treated for 1, 2, and 3 days, respectively, and the proliferative capacity of human skin cells was measured. As a result, Of the extracts were incubated for 2 days after treatment with human skin cells.
In addition, a concentration of 5 mu g / ml of juniper seed extract was treated with human skin cells and cultured for 2 days to analyze colony forming cells (CFU), and telomerase activity was evaluated. The CFU value was increased and telomerase activity was confirmed as compared with the untreated group, and it was confirmed that the extract of the fragrance of jukebang of the above condition is a substance for improving the proliferation of human skin cells.
In the present invention, the term "telomerase activity" is an in vivo enzyme comprising a protein as a main constituent. It contains an RNA molecule and functions to extend a telomeric sequence of a repeat sequence at the end of a chromosome . On the other hand, the telomerase gene is present in all cells, but most normal cells do not show activity, whereas cancer cells have about 90% of activity. The term "telomere" in the present invention means a base sequence region at the end of a chromosome. As the cell division progresses, the length of the telomere becomes shorter and eventually the cell replication is stopped. Therefore, .
Meanwhile, in the above embodiment of the present invention, the activation of telomerase by the extract of jellybean means that telomeres which cause senescence are elongated to inhibit aging of cells, thereby improving cell proliferation ability.
In one embodiment of the present invention, in order to confirm whether or not the myrtle extract that activates telomerase is a cancer cell, the proliferative ability of human skin cells was confirmed. As a result, it was confirmed that the proliferative capacity of human skin cells treated with the juniper extract was improved, but the growth rate was similar to that of the cells not treated with the juniper extract. In addition, it was confirmed that cell proliferation was restricted through intercellular contact, and it was confirmed that the extract of juniper jelly enhances cell proliferation ability and does not cause cancer cell formation.
In addition, in one embodiment of the present invention, when the extract of juniper seed extract at a concentration of 5 μg / ml is cultured for 2 days after human skin cells are cultured, the activity of human skin cells is increased, Respectively.
In the present invention, the human skin cells may be CCD-986SK cells, but the present invention is not limited thereto.
In the present invention, the term "CCD-986SK cell" is a type of human skin fibroblast cell, and has the shape of a fibroblast and a pattern showing a monolayer growing pattern. In one embodiment of the present invention, the CCD-986SK cells as human skin cells were cultured in a CO 2 incubator at 37 ° C using IMDM (Iscore's Modified Dulbecco's Medium) containing 10% FBS.
In another aspect, the present invention provides a method for improving the proliferation of human skin cells, which comprises treating a separated human skin cell with a bamboo extract.
In the present invention, the human skin cells may be CCD-986SK cells, but the present invention is not limited thereto. Meanwhile, the term "CCD-986SK cell" in the present invention is the same as that described above.
In the present invention, the jukebox extract may have a concentration of 1 / / ml to 7 / / ml, and the concentration may be 1 / / ml to 5 / / ml. In addition, the concentration of the fragrance extract may be 5 μg / ml.
In the present invention, the juniper extract may be treated with human skin cells for 1 to 3 days, but not limited thereto. Specifically, the juniper extract may be treated with human skin cells for 2 days.
The invention juniper (Juniperus chinensis extract as an active ingredient has the effect of activating telomerase of human skin cells and increasing the migration activity of human skin cells and improving the proliferation of human skin cells.
Fig. 1 is a graph showing cytotoxicity when human skin cells are treated with a juniper extract at different concentrations.
FIG. 2A is a graph showing the enhanced proliferative activity of cells when human skin cells are treated with a concentration of 5 / / ml of juniper extract.
FIG. 2B is a graph showing changes in the cell proliferative capacity with time after treatment of human skin cells with the extract of Leuca juncea by concentration. FIG.
Fig. 3 is a graph showing an increase in the value of the colony forming unit (CFU) as a result of culturing human skin cells at 5 mu g / ml concentration of juniper extract and culturing for 2 days.
Fig. 4 is a graph showing improvement in telomerase activity as a result of culturing human skin cells at a concentration of 5 mu g / ml of juniper extract and culturing for 2 days.
FIG. 5 is a graph showing the proliferative capacity of human skin cells as a result of culturing human skin cells at 5 mu g / ml concentration of juniper extract and culturing for 2 days.
FIG. 6 is a graph showing cell migration by in vitro transgenic plates after treatment of human skin cells with 5 / / ml of juniper extract at a concentration of 5 / / ml for 2 days.
Hereinafter, the constitution and effects of the present invention will be described in more detail with reference to Examples and Experimental Examples. These Examples and Experimental Examples are only for illustrating the present invention, and the scope of the present invention is not limited by these Examples and Experimental Examples.
Example 1: Preparation of juniper extract
In order to confirm the use of the improvement of the proliferation of the human skin cells (CCD-986SK) of the jelly extract, the jelly berry extract was prepared as follows.
Five kilograms of Juniperus chinensis were weighed and ground into powder and extracted with ethanol. Concretely, ethanol solvent five times as much as the sample was added and then extracted three times at 65 times. The extracted samples were filtered, concentrated by using a vacuum concentrator, and lyophilized to yield a weight.
Example 2: human Skin cell culture
Human skin cells (CCD-986SK) were cultured overnight at 37 ° C in a CO 2 incubator using IMDM (Iscore's Modified Dulbecco's Medium) containing 10% FBS. The medium was exchanged after the first two days and then every four days thereafter. After culturing for 2 days to 3 days, when the primary cultured cells reached a density of 70-80%, they were subcultured using 0.025% trypsin solution.
Example 3: Human of juniper extract To skin cells Cytotoxicity against cell cytotoxiticy ) Measure
Cell viability was measured using trypan blue exclusion in order to confirm the effect of the extract of the jellyfish obtained in Example 1 on the cell viability.
More specifically, to determine the cell toxicity of juniper extract, and inoculated with the human skin cells (CCD-986SK) cultured in Example 2 at a concentration of 5
As a result, when the concentration of juniper extract was 1 to 5 / / ml, the survival rate of the cells was increased as compared with the control group not treated with the juniper extract. On the other hand, the cell survival rate tends to be significantly decreased when the concentration of juniper extract is 10 / / ml, and when the concentration of juniper extract is 25 / / ml or more, the cell survival rate is dose- (Fig. 1).
Through the changes of human skin cell survival rate according to the concentration of the fragrant extract, it was confirmed that the cytotoxicity was different according to the treatment concentration of the fragrance extract. Specifically, when the extract concentration is 1 and 5 μg / ml, the extract is a safe extract without cytotoxicity, whereas when the extract concentration is 10 μg / ml, the cytotoxicity tends to appear, and furthermore, Was 25 μg / ml or more, it was confirmed that there was strong cytotoxicity. As a result, it was confirmed that the concentration of juniper extract was not cytotoxic within the range of 1 to 5 占 퐂 / ml and was safe for cells. Hereinafter, experiments for confirming optimal conditions for improving human skin cell proliferative ability and experiments for improving skin cell proliferating ability under the above conditions were conducted.
Example 4: Human according to the concentration of juniper extract Skin cell Proliferative ability Assessment and optimization
Human skin cells (CCD-986SK) were treated with 1, and 5 ㎍ / ml of juniper extract, which had no cytotoxicity as described in Example 3, and 10 / / ml of juniper extract, Cell proliferation was evaluated. Cell proliferation was assessed by visual cell count with trypan blue exclusion.
Specifically, the cultured human skin cells (CCD-986SK) of Example 2 were inoculated into a 10 cm dish at a density of 5 x 10 5 and stabilized with IMDM medium at 37 in a CO 2 incubator. Thereafter, the juniper extracts at the concentrations of 1, 5, and 10 / / ml were treated in the medium. In order to evaluate the cell proliferative activity after the treatment with the juniper extract, the juniper extract was treated, and the cells were cultured for 1 day, 2 days, and 3 days, respectively. In all proliferation assays, three wells were used for each condition and each experiment was repeated at least three times.
As a result, treatment with 1 and 5 μg / ml of juniper extract significantly increased the proliferation of human skin cells (CCD-986SK). When treated with 5 ug / ml of juniper extract, (Fig. 2a). In addition, as a result of treatment with the juniper seed extract according to the administration period, when the juniper extract of 5 / / ml was treated with human skin cells and cultured for 2 days, the growth ability was improved by 140% or more as compared with the control without the extract . On the other hand, when the extract was treated with 10 μg / ml of juniper seed extract, the proliferative activity of the skin cells was decreased in comparison with the control, and the proliferative activity of the skin cells when cultured for 3 days was 80% (Fig. 2B).
As a result of confirming the ability of the human skin cells to proliferate according to the concentration of the above-mentioned fragrance extract, it was confirmed that the human skin cell proliferating ability was improved when 1, and 5 ㎍ / ml of the fragrance extract was treated. On the other hand, it was confirmed that when the extract was treated with 10 / / ml of juniper seed extract, the proliferative capacity of human skin cells was decreased and the proliferative activity of human skin cells could be improved in the range of 1 to 5 ㎍ / ml of juniper extract Respectively. Furthermore, it was confirmed that conditions for treating human skin cells with a concentration of 5 / / ml of juniper seed extract and culturing for 2 days were optimal conditions for improving human skin cell proliferation ability.
Example 5: Leaf extract Skin cell Verification of proliferation improvement effect
Example 5-1: Optimal Under the conditions Colony Forming cell ( CFU ) Analysis
In an experiment for verifying the degree of improvement in the proliferation of human skin cells, the extract of the juniper seeds having the optimum conditions identified in Example 4 was treated with human skin cells (CCD-986SK) to evaluate the proliferation efficiency of the colony forming unit (CFU). Since CFU is a single cell population, it can be seen that the increase in CFU value can actively stimulate the growth of human skin cells (CCD-986SK).
Specifically, human skin cells (CCD-986SK) were inoculated in a 10 cm dish at 5 x 10 5 density and stabilized in IMDM containing 2% FBS for 8 hours at 37 ° C CO 2 incubator. After stabilization, a concentration of 5 / / ml bamboo extract was treated with human skin cells and cultured for 2 days. For CFU analysis, human skin cells are juniper extract untreated (CCD-986SK) and juniper extract is inoculated with human skin cells (CCD-986SK) processed in each 10cm dishes at a density of 2
As a result, human skin cells (CCD-986SK) cultured on human skin cells at a concentration of 5 μg / ml for 2 days and human skin cells (CCD-986SK) The value of CFU increased about 1.5 times (Fig. 3). Thus, it was confirmed that when the extract of juniper japonica at a concentration of 5 / / ml was treated with human skin cells and cultured for 2 days, it was effective in improving the proliferation of the cells.
Example 5-2: Telomerase activation( Telomerase activity
In another experiment for verifying the degree of improvement of proliferation of human skin cells, the telomerase activity of inhibiting cell senescence was confirmed.
Specifically, human skin cells (CCD-986SK) were inoculated in a 10 cm dish at 5 x 10 5 density and stabilized in IMDM containing 2% FBS for 8 hours at 37 ° C CO 2 incubator. After stabilization, the stabilized cells were treated with 5 / / ml of juniper extract and cultured for 2 days. For evaluation of telomerase activity, human skin cells (CCD-986SK), which had not been treated with the juniper extract, and human bark cells (CCD-986SK), which had been cultured for 2 days, And then telomerase activity was assessed using the Telomerase PCR ELISA kit (Roche Diagnostics).
As a result, human skin cells cultured for 2 days after treatment with 5 / / ml of juniper extract increased the telomerase activity of human skin cells by about 1.5-fold (Fig. 4).
An increase in telomerase activity is characteristic of cells with enhanced proliferation and decreased telomerase activity means that cells are differentiated. From the above experimental results, it was confirmed that the extract of juniper japonica at a concentration of 5 / / ml is effective in improving the proliferation of human skin cells by activating telomerase.
Example 6: Human caused by treatment of juniper extract Skin cell Extended Proliferative ability Confirm
As shown in the above Example 5-2, it was confirmed that the fragrance extract of oriental jelly was activating telomerase. Although telomerase gene is present in all cells, activity is not found in most normal cells, whereas about 90% of cancer cells are active, and the extract of jellyfish is a cancer cell (CCD-986SK) And confirmed the proliferative ability to prolong it.
As a result of the experiment, human skin cells not treated with jellyfish extract during the prolonged incubation period showed a gradual decrease in the proliferative capacity, and cells at the end of the proliferative life cycle were morphologically similar to aged fibroblasts, Respectively. On the other hand, in the case of human skin cells treated with 5 / / ml of juniper extract, the growth ability of the cells was improved, but the growth rate was similar to that of human skin cells not treated with the juniper extract. In addition, cell proliferation was restricted through intercellular contact (Fig. 5).
These results show that the improvement in the growth potential of human skin cells (CCD-986SK) treated with the juniper extract is not a change in the properties of cell growth. In other words, it means that the juniper extract only improves human skin cell proliferation ability and does not cancer cell.
Example 7: In vitro Transsegu Evaluation of Cell Migration by Plate
To determine whether human skin cells with improved proliferation (CCD-986SK) exhibited normal activity as a result of treatment of the jellyfish extract, the mobility activity of human skin cells treated with a juniper extract (5 / / ml, 2 days) was measured .
Specifically, human skin cells (CCD-986SK) were inoculated in a 10 cm dish at a concentration of 5 x 10 5 and stabilized in an IMDM medium containing 2% FBS for 8 hours in a CO 2 incubator under 37 conditions. After stabilization, a concentration of 5 / / ml bamboo extract was treated with human skin cells and cultured for 2 days. The cultured cells were transferred to a Costar transwell membrane (8 μm pore size) and placed in a 6-well plate. Below the membrane of each sphere was added IMDM medium containing juniper extract and 2% FBS. The cells in the upper chamber were incubated in IMDM medium containing 2% FBS for 2 hours at 37, and plates were incubated overnight in a CO 2 incubator at 37 ° C. Subsequently, the cells on the lower surface were dried, stained with Harris hematoxylin for 20 minutes and washed. The stained insert was placed on the objective slide and the number of cells on the lower surface was measured with an inverted bright field microscope at 200x magnification. The images were repeatedly measured at 10 magnifications on a microscope at a magnification of 200 times and the average value thereof was represented by data. The activity of cell migration was expressed as the number of cells per arbitrarily indicated spontaneous migration region.
As a result, the human skin cells (CCD-986SK) cultured for 2 days after treatment with 5 ㎍ / ml of juniper extract showed a time-dependent effect on human skin cells (CCD-986SK) Increased mobile efficiency. In addition, the activity of cell migration was increased by about 20% or more as compared with the human skin cells cultured for 1 day after the treatment of the fragrance extract (Fig. 6). Thus, it was confirmed that human skin cells were cultured for 2 days after treatment with 5 / / ml of juniper extract, and the activity of human skin cells was increased to exhibit normal cell activity.
From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.
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| CN107837208B (en) * | 2017-08-17 | 2021-02-02 | 上海联衡生物科技有限公司 | Preparation method of telomerase composition and application of telomerase composition in anti-aging and anti-retrogrowth cosmetics |
| KR102019819B1 (en) * | 2017-11-15 | 2019-09-09 | 부산대학교 산학협력단 | Composition for preventing or treating atopic dermatitis comprising cupressuflavone or amentoflavone |
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| 전혜지 et al, ‘노간주나무(Juniperus rigida Sieb.)의 주름개선 효과’, Korean Journal of Microbiology and Biotechnology, 2013, Vol. 41, No. 4, 449-455* |
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