KR20180134586A - Compositions for preventing skin damage and accelerating skin regeneration containing Allium cepa Extracts - Google Patents

Abstract

The present invention relates to a method for manufacturing a composition for preventing skin damage and promoting skin regeneration, and a composition manufactured thereby. The composition comprises onion extracted powder manufactured by a method comprising: a first step of immersing dried onions in ethanol to obtain an onion extract; a second step of filtering the onion extract; a third step of concentrating the filtered onion extract; a fourth step of dissolving the concentrated solution so as to have a solid content of 5-20 wt%; a fifth step of performing a first fractionation of the solution with hexane and then a second fractionation with ethyl acetate to remove non-polar components in the solution; and a sixth step of concentrating the fractions under reduced pressure to obtain onion extracted powder. The composition has an effect of preventing damage to the skin exposed to ultraviolet rays and promoting regeneration of the damaged skin while removing the odor of onions.

Description

Technical Field [0001] The present invention relates to a composition for preventing skin damage and promoting skin regeneration including onion extract, and a method for manufacturing the same. [0002] Compositions for preventing skin damage and accelerating skin regeneration containing Allium cepa Extracts

The present invention relates to a composition for prevention of skin damage and skin regeneration comprising an onion extract, and more particularly to a composition for preventing skin damage and improving skin, which comprises removing spe- cific onions and improving the content of spiraeoside contained in onion extracts, The present invention relates to a composition for prevention of skin damage and a composition for skin regeneration, which comprises an onion extract which prevents damage to skin and promotes regeneration of damaged skin, and a method for producing the same.

Photoaging, one of the main causes of skin aging, is responsible for most cosmetic and medical problems that occur on the skin.

This photoaging is rougher and more dry than the endogenous aging, and the skin elasticity is remarkably reduced, resulting in deeper, deep skin wrinkles and lesions such as sebaceous gland hyperplasia. The photoaged skin is slightly increased in thickness at first, then thinner than that of endogenous aging, and the shape and arrangement of keratinocytes becomes abnormal. The number of Langerhans cells is further reduced and the local immune function deteriorates severely. Melanocyte counts are decreased, but cell activity is increased irregularly, resulting in the appearance of a mixture of low pigmentation and hyperplasia with staining in the skin, and the likelihood of developing malignant tumors is markedly increased.

In addition, the photoaging skin has a much smaller amount of collagen in the dermis than the endogenous aging skin. Elastic bones, which account for 3-4% of the dermis, decrease in number and diameter, become shorter in length, and become calcified. Furthermore, in the photoaged epidermis, deformed elastotic material is widely deposited on the upper part of the dermis, and the decrease of hyaluronic acid in the mucopolysaccharide of the dermis substrate is remarkable, resulting in a decrease in skin tension. Decreased dermal thickness and changes in its constituents cause skin elasticity to be reduced, making it appear as an outwardly sagging or thick, wrinkled skin.

The wavelength range of sunlight that causes such photoaging is the ultraviolet region. Sunlight is mainly caused by ultraviolet B (UVB, 290-320 nm), which causes DNA damages in the skin and causes symptoms of photoaging.

Therefore, it is required to develop cosmetic raw materials that reduce DNA damage caused by ultraviolet rays. DNA-mediated mitochondria play an important role in apoptotic cell death and necrotic cell death. An early stage feature of programmed cell death is the disruption of activated mitochondria, which is a phenomenon such as changes in basement membrane potential and changes in the oxidation-reduction potential of mitochondria. Changes in the mitochondrial transmembrane potential of this early apoptosis phenomenon can be measured using a fluorochrome sensitive to the electrochemical potential inside the mitochondria. The green fluorescent cyanine dye JC-1, a green fluorescent cyanine dye, is the most widely used dye. It penetrates into the cytosol of eukaryotic cells and accumulates in mitochondria according to dislocation. Upon accumulation, JC-1 undergoes a fluorescence emission shift from green (~ 529 nm) to red (~ 590 nm), resulting in a decrease in red / green fluorescence intensity ratio when mitochondrial polarization occurs.

Moreover, the outer skin cells, which are the outer part of the skin, are constantly peeled off, and other cells growing beneath it continue to take their place. Therefore, when skin damage is caused by external stimuli, wound healing occurs immediately. Although the basement membrane is exposed due to the damage of the epidermis and dermis, the wound on which the epidermis is lost has a scarlet color on the wound surface.

The formation of new connective tissue in damaged skin is a new process of angiogenesis and collagen synthesis. In the process of collagen synthesis, fibroblasts play an important role in the wound repair process as they are the most important cells in collagen synthesis and other connective tissue formation. Fibroblasts move and proliferate in response to products produced by inflammation and growth factors such as platelets and macrophages, and begin to appear on the wound at the end of the inflammation. The wound healing by the primary healing process can fill the injured area with only a small amount of collagen, and collagen synthesis occurs almost simultaneously with epidermal cell migration.

The present applicant has made a certain starting point by a cell scratch assay and then conducted an experiment to microscopically observe how the cells grow over time after the sample treatment according to the present invention, It has developed an onion extract that prevents skin damage and promotes regeneration of damaged skin.

Korean Patent Publication No. 10-2004-0012913

Accordingly, an object of the present invention is to provide an onion extract which prevents the damage of skin exposed to ultraviolet rays and promotes the regeneration of damaged skin by improving the content of spiraeoside contained in onion extract, And a composition for preventing skin damage and regenerating the skin.

In order to solve the above-mentioned problems, the present invention provides a method for preventing skin damage and promoting skin regeneration,

A first step of immersing the dried onion in ethanol to obtain an onion extract; A second step of filtering the onion extract; A third step of concentrating the filtered onion extract; A fourth step of dissolving the concentrated liquid so as to have a solid content of 5 to 20% by weight; And a fifth step of fractionating the solution with hexane followed by a second fractionation with ethyl acetate to remove non-polar components in the solution.

In addition, the present invention provides a method for preventing skin damage and promoting skin regeneration,

And concentrating the fractions under reduced pressure to obtain an onion-extracted powder.

Here, the step of obtaining the onion extract of the first step comprises:

Dried onions are extracted by immersion in 95% ethanol at room temperature for 1-5 days at a ratio of onion: ethanol = 1: 5% by weight.

Further, the filtration in the second step is characterized by extracting with a filter having a mesh size of 0.1 to 10 mu m.

The concentration of the third step is characterized in that the onion extract is prepared by concentration at a temperature of 40 to 70 ° C.

In the sixth step, the fraction is reduced in pressure at a temperature of 40 to 100 ° C to 40 to 550 Torr and concentrated to obtain an onion-extracted powder.

In addition, the onion extract powder prepared by the method for preventing skin damage and promoting skin regeneration according to the present invention is characterized in that the content of spearoside is 10.0% by weight to 40.0% by weight in addition to the effect of improving odor.

The present invention also provides cosmetics containing the composition for preventing skin damage and promoting skin regeneration according to the present invention.

In addition, the cosmetic for preventing skin damage and promoting skin regeneration according to the present invention is characterized in that the onion-extracted powder of the composition is dissolved in 10 to 40% BG.

The composition for preventing skin damage and promoting skin regeneration according to the present invention can be applied to existing products in addition to essence, lotion, cream, wash-off, or leave-on products according to the content or use method. It is possible to use it as an additive of a moisturizing and moisturizing cosmetic composition which can be applied in another category that can be given.

The cosmetics according to the present invention can be prepared in any conventional formulation as well as in lotion or lip make-up. Additives such as a softening agent, a flavoring, a pigment, an antioxidant, a pH adjuster, etc., Of course it is.

The onion extracts provided by the present invention have the effect of preventing damage to the skin exposed to ultraviolet rays while promoting the regeneration of damaged skin while removing the odor of the onion.

1 is a graph showing the toxicity test results of the onion-extracted powder of the first embodiment produced according to the present invention,
FIG. 2 is a graph showing the toxicity test results of spireoside, an indicator material corresponding to FIG. 1,
FIG. 3 is a photograph showing the comparison of the degree of dyeing of the onion extract powder prepared according to the present invention and the indicator substance, spireoside, in comparison with the control group and the UVB 50 mJ treatment group,
4 is a comparative graph showing the toxicity test results of the onion-extracted powder and the spioroside of Example 2,
5 to 15 are graphs showing the effect of promoting cell regeneration of the onion extract powder and the indicator substance, spireoside. Fig. 5 is control, Fig. 6 is FBS 2%, Fig. 7 is onion extract powder 25 占 퐂 / Fig. 10 shows an onion extract powder of 250 g / ml, Fig. 11 shows a sporeoside of 0.15 g / ml, Fig. 12 shows a mixture of sporeoside 0.3 Fig. 13 shows the result of treatment with 0.6 / / ml of spearoside, Fig. 14 shows 1.5 / / ml of spearoside, and Fig. 15 shows treatment with 3 / / ml of spearoside.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the drawings, examples and experimental examples. It is needless to say that the following drawings, examples and experimental examples are illustrative of the present invention, but the content of the present invention is not limited thereto. In addition, a description of a conventional measurement experiment in an experimental procedure for measuring efficacy is omitted.

1. Experimental Evaluation Method of Photovoltaic Effectiveness

1-1. Cytotoxicity assay (MTT assay)

In order to examine the photoaging effect of onion extracts, 24-well plates were inoculated with horny cells (HaCaT) for 24 hours, the culture medium was replaced with serum free medium, Lt; / RTI > The supernatant was removed, and 1 ml of DMSO was added to dissolve the MTT-formazan crystals. The absorbance at 570 nm was measured with an ELISA reader. . Cytotoxicity is expressed as a percentage of absorbance compared to the control.

1-2. JC-1 staining (JC-1 staining)

To test the changes in mitochondrial membrane potential, keratinocytes were plated on a 35 mm plate, stabilized for 24 hours, replaced with serum-free medium, and treated. After 1 hour, wash with HBSS (Hank's Balanced Salt solution) buffer, add fresh HBSS, and irradiate with UVB 50 mJ / ㎠. After replacing the medium with serum-free medium, the sample is treated and incubated at 37 ° C in a 5% CO ₂ incubator for 24 hours. After removal of the medium, treatment with 1 μg / ml JC-1 solution is carried out at 37 ° C in a 5% CO ₂ incubator for 2 hours without light. The medium is removed and observed under a fluorescence microscope.

1-3. Equipment and reagents used

(1) ELISA reader: Infinite M200 (Tecan, Austria), (2) MTT: Thiazolyl Blue Tetrazolium Bromide (M5655, Sigma-Aldrich) ⑤ JC-1 dye (Invitrogen), ⑦ Leica Microscope (Applied Biosystems, USA), ⑤ Real-Time PCR: 7500 Fast with Power SYBR Green PCR master mix

1-4. Method of preparing sample of extractive onion extract powder and method of analyzing content of spireoside which is an index substance in extract

Samples were prepared and analyzed in the following manner to verify the improvement of the onion extract and to analyze the content of spioroside in the extract.

1-5. Sample preparation

Onions to be used for onion extract are selected, washed and dried.

Then, the onion extract is obtained by immersing the dried onion at 95% ethanol in an amount of 1: 5, preferably 1: 2, by weight, preferably 1: 5, preferably 2 days at room temperature .

Next, the onion extract is filtered with a filter having a mesh size of 0.1 to 10 μm, preferably 1 μm.

The filtered onion extract is concentrated to a concentrate at a temperature of approximately 40 to 70 DEG C, preferably 60 DEG C.

The prepared concentrated liquid is dissolved in purified water to have a solid content of 5 to 20% by weight, preferably 10% by weight, to prepare a dissolution liquid.

The prepared dissolving solution is fractionated with hexane 1 to 6 times, preferably 3 times or more, to remove volatile components, which are known non-polar components in the solution of the onion extract, and then diluted with ethyl acetate 1 to 4 times, The non-polar component remaining in the solution of the onion extract is removed by fractionation two or more times so that the extraction amount of the spireoside in the total sample content is increased.

The fractions obtained by fractionation with ethyl acetate were concentrated under reduced pressure at a temperature of approximately 40 to 100 ° C, preferably 50 to 60 ° C, gauzing, preferably 55 ° C, to approximately 40 to 550 Torr, to obtain an onion-extracted powder.

1-6. Manufacture of the indicator substance spireoside

As a comparative sample, 1000 ppm of spirososide as an indicator material was dissolved in 30% BG (butylene glycol) by heating, and the filtrate was filtered with a filter having a mesh size of 0.1 to 0.3 탆, preferably 0.22 탆 .

A solution of 2.1 mg of the filtered spearoside in a 50-mL volumetric flask containing MeOH is used as a standard solution.

In addition, 50 mg of the onion extract powder is completely dissolved in MeOH and filtered to 0.45 쨉 m as a test sample.

Here, the content analysis conditions are as follows.

 Column: Capcell Pak C18 4.6 x 250 mm (Shiseido HPLC column)

 Flow rate: 1.0 mL / min

 Detection: UV 280 nm

 Temp: 30 ° C

 Injection volume: 20 Ul

 Analysis time: 30 min

Mobile phase: ACN, purified water, acetic acid (250: 750: 1)

1-7. Experiment result

The onion extract powder obtained by ethanol extraction, the onion extract powder obtained by fractionation with ethyl acetate, and the fraction obtained by the first fractionation with hexane and the second fractionation with ethyl acetate, 1.

sample Spearoside content
(ppm) 95% EtOH powder 4,500 ppm
=> 0.45% The second fraction powder
(Improved content) 112,000 ppm
=> 11.2% The first and second fraction powders
(Improvement of content, improvement of intake) 358,000 ppm
=> 35.8%

As shown in Table 1, in the case of powder extracted with 95% EtOH, the content of spireoside was 0.45% and had a characteristic onion. In the case of the onion extract powder fractionated second with ethyl acetate, the content of spioroside was 11.2%, and the odor was not improved much. On the other hand, after the first fractionation with hexane, the second fraction of the ethyl acetate fraction showed the highest effective ingredient value with the improvement of the pore size and the content of spireoside of 35.8%.

When the onion-extracted powder thus produced is used in cosmetics, the use of the powder-type raw material may not be easy depending on the formulation, and thus the powder-removed powder is processed into a liquid form.

For this purpose, the onion extract powder with improved odor was dissolved in 10-40% BG, preferably 30% butylene glycol (BG) and hexanediol, respectively, and the results are shown in Table 2 below.

sample Spearoside content
(ppm) The first and second fraction powders 358,000 ppm
=> 35.8% The first and second fraction powders
30% BG-solution type 1,030ppm
=> 0.103% The first and second fraction powders
Hexanediol-solution type 90ppm
=> 0.009%

As shown in Table 2, it was confirmed that about 30% BG dissolved spireoside was detected about 11 times higher than when dissolved in Hexanediol.

Therefore, it is considered that onion extract powder improved by the formulation of cosmetics or the product in which the powder is dissolved in 30% BG can be used.

The following tests were carried out using onion extract powder, which has improved odor, as a sample.

1-7-1. Cytotoxicity Test (MTT Assay)

Prior to JC-1 staining, toxicity was assessed using keratinocytes (HaCaT). The experimental results are shown in Table 3 and FIGS. 1 and 2.

As indicated in Table 3 and Figures 1 and 2, no toxicity was detected at the whole concentration of the onion extract powder. Therefore, the experiment was carried out with concentrations of onion extract powder of 50, 100, 250 and 500 ㎍ / ㎖, and the surface element, spireoside, was added to the concentration of 0.3, 0.6, 1.5 and 3 ㎍ / The experiment was carried out.

1-7-2. JC-1 staining

The degree of staining of onion extract powder and the indicator substance, spearoside, compared with control (untreated) and UVB 50 mJ treated group, which is a control group, is shown in a comparative photograph in FIG.

As shown in Fig. 3, red fluorescence staining in living cells seems to be increased at a total concentration than in the UVB-treated group, and high light protection effect was observed at a lower concentration than at a high concentration.

Therefore, it is considered that onion extract powder and spearoside are highly effective in inhibiting UVB - induced apoptosis at a lower concentration than high concentration.

2. Evaluation method of wound healing efficacy

2-1. Cytotoxicity assay (MTT assay)

In order to examine the wound healing efficacy of promoting skin regeneration of onion extract, 24-well plate was inoculated with human fibroblast and cultured for 24 hours. Then, the culture medium was replaced with serum-free medium The samples are treated for each concentration and cultured for 24 hours. The supernatant was removed, and 1 ml of DMSO was added to dissolve the MTT-formazan crystals. The absorbance at 570 nm was measured with an ELISA reader. . Cytotoxicity is expressed as a percentage of absorbance compared to the control.

2-2. Wound healing assay

Fibroblast is inoculated on a 60 mm plate and cultured for 24 hours. After incubation, scrape the sample linearly and replace the sample with serum-free medium. After culturing for 3 or 6 days, photograph the cell with a microscope (Leica).

2-3. Equipment and reagents used

1) ELISA reader: Infinite M200 (Tecan, Austria), 2) MTT: Thiazolyl Blue Tetrazolium Bromide (M5655, Sigma-Aldrich), 3) Scraper (NUNC)

2-4. Sample information

The onion extract powder was prepared by the above-described evaluation of photoengraving efficacy, and dimethylsulfoxide (DMSO) was used as a solvent.

Spearoside was purchased from EXTRASYNTHESE, and dimethylsulfoxide (DMSO) was used as a solvent.

2-5. Test results

2-5-1. Cytotoxicity Test (MTT Assay)

The toxicity test results for onion extract powder and spioroside are shown in Table 4 and FIG. 4 below.

As shown in Table 4 and FIG. 4, no toxicity was detected at the whole concentration of onion extract powder and spearoside, and the concentration of onion extract powder 25, 50, 100, 250, 500 / / The migration test was carried out at 0.15, 0.5, 0.6, 1.5, and 3 ug / ml of spearoside.

2-5-2. Cell migration test

In order to observe the cell regeneration promoting activity of the onion extract powder and the indicator substance spearoside, a wound healing assay was carried out, and the results were divided by the concentration, Day 1, Day 2, Day 3, Day 4 and Day 7, Respectively.

FIG. 5 shows the results of the control, FIG. 6 shows FBS (Fetal bovine serum) treated with 2%, FIG. 7 shows onion extract powder of 25 .mu.g / Onion extract powder of 100 占 퐂 / ml, Onion extract powder of 250 占 퐂 / ml, Fig. 11: Sporeoside 0.15 占 퐂 / ml, Fig. 12: Sporeoside 0.3 占 퐂 / Ml, Fig. 14 shows the treatment with 1.5 占 퐂 / ml of the spioroside, and Fig. 15 shows the treatment with 3 占 퐂 / ml of the spioroside.

As shown in FIG. 5 to FIG. 16, the migration efficacy of the spioroside was observed in a concentration dependent manner, and the onion extract powder showed a total migration effect. In particular, at a concentration of 50 and 100 μg / %, Similar to that of the control.

Therefore, it is considered that onion extract powder and spearoside have the effect of promoting cell regeneration when a wound occurs.

Although the present invention has been described in connection with the preferred embodiments set forth above, it will be appreciated that various other modifications and variations can be made without departing from the spirit and scope of the invention, And variations.

Claims (9)

A first step of immersing the dried onion in ethanol to obtain an onion extract;
A second step of filtering the onion extract;
A third step of concentrating the filtered onion extract;
A fourth step of dissolving the concentrated liquid so as to have a solid content of 5 to 20% by weight; And
A first step of fractionating the solution with hexane followed by a second fractionation with ethyl acetate to remove a non-polar component in the solution; and a method for manufacturing a composition for promoting skin damage prevention and skin regeneration comprising an onion extract .
The method according to claim 1,
After the fifth step,
And concentrating the fractions under reduced pressure to obtain an onion-extracted powder. The method for preventing skin damage and promoting skin regeneration according to claim 1, further comprising:
The method according to claim 1,
The onion extract of the first step is obtained,
And extracting the dried onion with 95% ethanol at a ratio of onion: ethanol = 1: 5% by weight for 1 to 5 days at room temperature to thereby prevent skin damage and promote skin regeneration.
The method according to claim 1,
In the second step,
And extracting with a filter having a mesh size of 0.1 to 10 탆.
The method according to claim 1,
In the third step,
Wherein the onion extract is concentrated at a temperature of 40 to 70 ° C.
3. The method of claim 2,
In the sixth step,
Wherein the fraction is depressurized at a temperature of 40 to 100 DEG C to 40 to 550 Torr and concentrated to obtain an onion-extracted powder.
The onion extract according to any one of claims 1 to 6, wherein the onion extract has a pore-reducing effect and a poreose content of 10.0% by weight to 40.0% by weight. Gt;
6. A cosmetic comprising the onion extract prepared by the method of any one of claims 1 to 6, and a composition for promoting prevention of skin damage and promoting skin regeneration.
9. The method of claim 8,
Wherein the onion extract of the composition is an onion extract powder dissolved in 10 to 40% BG.

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