CN109010203B

CN109010203B - Composition for preventing skin damage and promoting skin regeneration and preparation method thereof

Info

Publication number: CN109010203B
Application number: CN201810086664.2A
Authority: CN
Inventors: 南世熙; 李度妍; 徐在龙; 朴晟希; 赵显垡; 辛容圭
Original assignee: Elsevier Brand Corp; Cosmecca Korea Co Ltd
Current assignee: Elsevier Brand Corp; Cosmecca Korea Co Ltd
Priority date: 2017-06-09
Filing date: 2018-01-30
Publication date: 2022-03-08
Anticipated expiration: 2038-01-30
Also published as: CN109010203A; KR102106813B1; KR20180134586A

Abstract

The present invention relates to a composition for preventing skin damage and promoting skin regeneration and a method for producing the same. The method comprises the following steps: stage 1 of immersing dried onion in ethanol to obtain onion extract; stage 2 of filtering the onion extract; stage 3 of concentrating the filtered onion extract; dissolving the solid content in the concentrated solution to 5-20 wt% in the 4 th stage; a 5 th stage of removing the nonpolar component in the solution after extracting the solution with hexane and ethyl acetate in sequence; stage 6, concentrating the extract under reduced pressure to obtain onion extract powder; and has effects of removing peculiar smell of onion, preventing ultraviolet skin injury and promoting damaged skin regeneration.

Description

Composition for preventing skin damage and promoting skin regeneration and preparation method thereof

Technical Field

The invention relates to a composition for preventing skin damage and promoting skin regeneration, in particular to a composition for preventing skin damage and promoting skin regeneration of an onion extract, which removes the peculiar smell of onion and improves the spiraeoside content in the onion extract so as to achieve the purposes of preventing skin damage and promoting the regeneration of damaged skin, and a preparation method thereof.

Background

Photoarradiation, the primary cause of skin aging, is becoming the source of most cosmetic and medical problems in skin.

Compared with intrinsic skin aging, the skin is rougher and drier under the condition of light radiation skin aging, and the skin elasticity is obviously reduced, so that the symptoms of skin sagging, deeper wrinkles, purpura, sebaceous gland hyperplasia and the like appear. After the thickness of the epidermis of the skin, which forms the skin aging by light irradiation, is initially slightly increased, it becomes thinner than the intrinsic skin aging, and the morphology and arrangement of glial cells become abnormal. The number of langerhans cells is also significantly reduced, resulting in a severe reduction of local immune function. The number of the melanocytes is also reduced, and the activity of the melanocytes is increased irregularly, so that the skin has the symptoms of speckle-like pigment reduction and generalized pigment abnormality, and the incidence rate of malignant tumors is also obviously improved.

Furthermore, the amount of glue in the dermal matrix of the skin forming photoradiation skin ageing is also significantly reduced compared to intrinsic skin ageing. When the quantity and the diameter of the elastin contained in the dermis are reduced by 3-4% and the length is shortened, intracutaneous calcareous deposition is caused, and in addition, a large area of elastic fiber substance (elastic material) is deposited in the upper layer of the dermis forming light radiation skin aging, hyaluronic acid in mucopolysaccharide (mucopolysaccharide) of a dermal matrix is remarkably reduced, so that the elasticity of the skin is reduced. The reduced thickness of the dermis and its compositional changes will result in a decrease in skin elasticity, resulting in significant sagging or wrinkling of the skin.

The wavelengths of light that cause photo-radiation skin aging are in the ultraviolet region. The main causes of sunburn are ultraviolet B (UVB, 290-320nm), which causes DNA damage in the skin and a series of symptoms of photo-irradiated skin aging.

Thus, there is a need for developing a cosmetic material capable of reducing DNA damage, and mitochondria containing DNA play an important role in apoptotic cell (apoptotic cell) death and necrotic cell (necrotic cell) death of nerve cells. The initial stage of programmed cell death is characterized by breakdown of activated mitochondria, which leads to changes in basal membrane potential and oxidation-reduction potential. The change in mitochondrial membrane potential (mitoc horizontal transmembrane potential) of this initial apoptosis (apoptosis) phenomenon can be measured using fluorochrome (fluorochrome) sensitive to the electrochemical potential (intracellular) inside the mitochondria, and the most commonly used green fluorochrome (green fluorescent cyanine dye) JC-1 is injected into the cytosol of eukaryotic cells (eukaryotic cells) and accumulates in the mitochondria along with the potential. The fluorescence emission shift of JC-1 on accumulation varies from green (-529 nm) to red (-590 nm), eventually polarizing the mitochondria and resulting in a decrease in the red/green fluorescence intensity ratio.

While the outermost skin cells of the skin are continually stripped and removed and replaced by new cells. Therefore, when the skin is damaged by external stimulation, the wound is healed immediately, and the damaged wound surface of the epidermis becomes bright red due to the exposure of the basement membrane.

The process of forming new connective tissue in damaged skin includes neovascularization and collagen synthesis. Fibroblasts (fibroplasts) during collagen synthesis (collagen synthesis) are the most important cells in collagen synthesis and other connective tissue formation, and play an important role in wound healing. Fibroblasts react to, move and proliferate to products and growth factors generated from platelets, macrophages, etc. during the inflammatory phase, and begin to appear at the wound site during the post-inflammatory phase. Wound healing in a one-time healing mode can fill the damaged part with only a small amount of collagen, and collagen synthesis and epidermal cell movement almost simultaneously occur.

In contrast, the present applicant developed an onion extract for preventing uv skin damage and promoting the regeneration of damaged skin by treating the reagent prepared in the present invention after preparing a certain starting point through a cell scratch test (cell scratch assay) and performing an experiment of observing cell growth with a microscope with time.

Patent document

Korean laid-open patent No. 10-2004-0012913

Disclosure of Invention

The present invention aims to provide an onion extract which removes the peculiar smell of onions and improves the content of spiraeoside in the onion extract, thereby preventing skin damage caused by ultraviolet rays and promoting skin regeneration, and a composition for preventing skin damage and promoting skin regeneration containing the same.

In order to solve the above problems, the method for producing a composition for preventing skin damage and promoting skin regeneration according to the present invention comprises:

stage 1 of immersing dried onion in ethanol to obtain onion extract; stage 2 of filtering the onion extract; stage 3 of concentrating the filtered onion extract; dissolving the solid content in the concentrated solution to 5-20 wt% in the 4 th stage; a 5 th stage of removing the nonpolar component in the solution after extracting the solution with hexane and ethyl acetate in sequence; and onion extracts obtained therefrom.

In addition, the method for producing the composition for preventing skin damage and promoting skin regeneration, which is provided by the present invention,

stage 6, which includes making onion extract powder by making the extract undergo the process of reduced pressure concentration; is characterized in that.

And, in the process of obtaining the onion extract of the above stage 1,

the onion is characterized in that dried onion and ethanol are soaked in 95% ethanol for 1-5 days at normal temperature according to the proportion of 1:5 wt% and then extracted.

Meanwhile, the filtration process of the 2 nd stage is characterized in that the extraction is carried out by using a filter with the mesh size of 0.1-10 μm.

Meanwhile, the concentration process of the 3 rd stage is characterized in that the onion extract is concentrated at the temperature of 40-70 ℃.

And, in the stage 6, concentrating the extract at 40-100 deg.C under reduced pressure of 40-550 Torr to obtain Bulbus Allii Cepae extract powder.

Meanwhile, the onion extract powder prepared by the method for preparing the composition for preventing skin damage and promoting skin regeneration according to the present invention has the deodorizing effect and the spiraea glycoside content of 10.0 wt% to 40.0 wt%.

Also, a cosmetic containing the composition for preventing skin damage and promoting skin regeneration prepared according to the present invention is provided.

Meanwhile, the cosmetic for preventing skin damage and promoting skin regeneration prepared according to the invention is characterized in that onion extract powder in the composition is dissolved by 10-40% BG.

The composition for preventing skin damage and promoting skin regeneration prepared according to the present invention can be used as a cosmetic raw material for moisturizing and maintaining moisture in essence, lotion, cream, Wash-Off or Leave-On products and other products, depending On the content or the use method thereof.

The cosmetic applicable to the present invention may be formulated into emulsion, lip care product or other conventional dosage forms, and any appropriate amount of general softener or additives such as perfume, pigment, antioxidant, pH regulator, etc. may be used in each dosage form.

The onion extract prepared according to the present invention has effects of preventing skin damage caused by ultraviolet rays and promoting regeneration of damaged skin while removing peculiar smell of onion.

Drawings

FIG. 1 is a graph showing the results of toxicity test of the onion extract powder according to the present invention;

FIG. 2 is a graph showing the results of toxicity tests of the reference substance spiraea glycoside corresponding to FIG. 1;

FIG. 3 is a graphic data comparing the degree of staining of onion extract powder prepared according to the present invention with control and UVB 50 mJ;

FIG. 4 is a graph comparing the toxicity test results of onion extract powder and meadowfoam;

FIGS. 5 to 15 are observation data of cell regeneration promoting effects of onion extract powder and embroidery glycoside as an index material, wherein FIG. 5 is control, FIG. 6 is FBS 2%, FIG. 7 is onion extract powder 25. mu.g/ml, FIG. 8 is onion extract powder 50. mu.g/ml, FIG. 9 is onion extract powder 100. mu.g/ml, FIG. 10 is onion extract powder 250. mu.g/ml, FIG. 11 is embroidery glycoside 0.15. mu.g/ml, FIG. 12 is embroidery glycoside 0.3. mu.g/ml, FIG. 13 is embroidery glycoside 0.6. mu.g/ml, FIG. 14 is embroidery glycoside 1.5. mu.g/ml, and FIG. 15 is a treatment case of embroidery glycoside 3. mu.g/ml.

Detailed Description

The invention will be explained in detail below with reference to the following figures and examples of implementation and experiments. The following drawings, examples and experimental examples are merely illustrative of the present invention and do not represent the contents of the present invention to be limited thereto. In addition, descriptions of commonly used measurement experiments during the efficacy testing experiments are omitted.

1. Experimental method for evaluating anti-light radiation skin aging effect

1-1. cytotoxicity assay (MTT assay)

To verify the anti-photo-irradiation skin aging effect of onion extract, after inoculating keratinocyte (HaCaT) in a 24-well plate and culturing for 24 hours, the cultured cell culture medium was replaced with serum free and then treated with different concentrations of reagents and cultured for 24 hours. After the culture medium in which the culture was completed was replaced with a 10-fold diluted culture medium using 2.5mg/ml MTT solution and the reaction was induced, the supernatant was removed and 1ml of DMSO was added, absorbance was measured at 570nm using an ELISA reader after the generated MTT-formazan crystals were dissolved, and the absorbance was expressed as a percentage after comparing cytotoxicity with a control group.

1-2.JC-1 dyeing

To verify the change in cell membrane potential of mitochondria, keratinocytes (Human keratincytes) were injected into 35mm plates, stabilized for 24 hours, and then replaced with serum-free medium to obtain samples. After 1 hour, the cells were washed with HBSS (Hank's Balanced Salt solution) buffer and observed for UVB 50mJ/cm2 after addition of fresh HBSS. Reagent treatment after medium replacement with serum-free medium and 5% CO at 37 deg.C₂Culturing for 24 hr in incubator, removing culture medium, treating with 1 μ g/ml JC-1 solution at 37 deg.C and 5% CO₂After 2 hours of the reaction in the incubator in the absence of light, the medium was removed and observed with a fluorescence microscope.

1-3. instruments and reagents used

(ii) ELISA reader Infinite M200(Tecan, Austria), MTT thiazole Blue Tetrazolium Bromide (Thiazolyl Blue Tetrazolium Bromide) (M5655, Sigma-Aldrich), RNA quantitation: Qubit fluorometer, with RNA BR test kit (Invitrogen, USA), cDNA Synthesis: high capacity RNA-cDNA kit (Applied Biosystems, USA), real-time PCR:7500 Rapid, with Power SYBR Green PCR Master mix (Applied Biosystems, USA), JC-1 dye (Invitrogen), and prism microscope

1-4. preparation method of onion extract powder reagent for improving odor and method for analyzing content of spiraea glycosides as index substance in extract

To verify the improvement of odor of the onion extract and to analyze the content of spiraea glycosides in the extract, reagent preparation and analysis were performed by the following methods.

1-5 preparation of reagents

Screening onion, cleaning and drying.

Soaking dried Bulbus Allii Cepae in 95% ethanol at a ratio of 1:5, preferably 1:2 wt% at room temperature for 1-5 days, preferably 2 days to obtain Bulbus Allii Cepae extract.

Then, the onion extract is filtered through a filter having a mesh size of 0.1 to 10 μm or preferably 1 μm.

Concentrating the filtered onion extract at about 40-70 deg.C, preferably 60 deg.C to obtain a concentrated solution.

And dissolving the prepared concentrated solution with purified water to obtain a dissolved solution with the solid content of 5-20 wt%, preferably 10 wt%.

The resultant solution is extracted with hexane 1 to 6 times, preferably 3 times or more to remove volatile components of nonpolar components generating odor in the onion extract solution, and then the remaining nonpolar components in the solution are extracted with ethyl acetate 1 to 4 times, preferably 2 times or more to increase the extraction content of spiraeoside in the total reagent content.

The extract is obtained by extracting with ethyl acetate as above, and concentrating at about 40-100 deg.C, preferably 50-60 deg.C, and most preferably 55 deg.C under reduced pressure of about 40-550 Torr to obtain onion extract powder.

1-6. preparation of the marker substance spiraea glycoside

Further, as a comparative reagent, 1,000ppm of the marker substance spiraea glycoside was dissolved by heating with 30% BG (butylene glycol), and then filtered with a filter having a mesh size of 0.1 to 0.3 μm or preferably 0.22 μm.

After the filtered spiraea glycoside (2.1 mg) was put into a 50mL flask, MeOH was added and dissolved, and the solution was used as a standard solution.

In addition, 50mg of onion extract powder was completely dissolved in MeOH, and the resulting solution was filtered with 0.45 μm to obtain a sample.

At this time, the content analysis conditions were as follows:

column: capcell Pak C184.6 x 250mm (Shiseido HPLC column)

Flow rate: 1.0mL/min

And (3) detection: UV 280nm

Temperature: 30 deg.C

Injection volume 20Ul

Analysis time: 30min

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

1-7. Experimental results

The results of the experiment on the spiraea glycoside content of the onion extract powder obtained by ethanol extraction, the onion extract powder obtained by ethyl acetate extraction, and the onion extract powder obtained by primary extraction with hexane and secondary extraction with ethyl acetate are shown in table 1.

[ TABLE 1 ]

As shown in table 1, the powder extracted with 95% EtOH had an spiraea glycoside content of 0.45% and had a characteristic odor of onions. The powder of onion extract extracted 2 times with ethyl acetate had an spiraea glycoside content of 11.2% and the odor was not much improved. On the contrary, the powder obtained by extracting with hexane for 1 time and then with ethyl acetate for 2 times has completely improved odor, and contains spiraea glycoside up to 35.8%, and has high effective component value.

When the onion extract powder thus prepared was used in cosmetics, it was possible that powdery raw materials could not be easily used depending on the formulation, and thus experiments were conducted in which odorless powder was processed in a liquid state.

For this purpose, the onion extract powder with improved odor was dissolved with 10 to 40% BG, preferably 30% Butylene Glycol (BG) and hexylene glycol (Hexanediol), respectively, and the results are shown in table 2.

[ TABLE 2 ]

As shown in table 2, the value of meadowfoam obtained by dissolution with 30% BG was about 11 times higher than when dissolved with hexylene glycol.

Thus, an onion extract powder with improved odor or a product in which the powder is dissolved with 30% BG can be used according to the cosmetic formulation.

The odor-improving onion extract powder was used as a reagent to carry out the following tests.

1-7-1. cytotoxicity Assay (MTT Assay)

Toxicity evaluation was performed using keratinocytes (HaCaT) before JC-1 staining, and the results are shown in Table 3, FIGS. 1 and 2.

As shown in Table 3, FIGS. 1 and 2, since no toxicity was detected in the concentration of the onion extract powder, experiments were carried out with onion extract powders having different concentrations of 50, 100, 250, 500. mu.g/ml, etc., while experiments with different concentrations of 0.3, 0.6, 1.5, 3. mu.g/ml, etc., were carried out with the meadowfoam according to the content in the onion extract.

[ TABLE 3 ]

1-7-2.JC-1 dyeing

The results of comparing the staining degrees of the onion extract powder with those of the control group (untreated) and the control group treated with UVB 50mJ are shown in the comparison chart of FIG. 3.

As shown in fig. 3, the number of red fluorescent stains of living cells was increased in all concentrations compared to the UVB-treated group, and the photoprotective efficacy was greater at low concentrations than at high concentrations.

It can be judged from this that the onion extract powder has more excellent efficacy against cell destruction by UVB at low concentrations.

2. Experimental method for evaluating wound healing efficacy

2-1 evaluation of cytotoxicity (MTT assay)

To verify the skin regeneration and wound healing effects of onion extract, fibroblast (human fibroblast) cells were seeded on a 24-well plate and cultured for 24 hours, and the cell culture medium was replaced with a serum-free medium, treated at each reagent concentration, and cultured for 24 hours. After the culture medium of the cells which have completed the culture was replaced with a 10-fold diluted medium of 2.5mg/ml MTT solution and the reaction was induced, the supernatant was removed and 1ml of DMSO was added, absorbance was measured at 570nm with an ELISA reader after the generated MTT-formazan crystals were dissolved, and the absorbance was expressed as a percentage after comparing cytotoxicity with a control group.

2-2. wound healing test

After inoculation of the Fibrolast in 60mm plates for 24 hours, the plates were scraped straight and replaced with serum-free medium after completion of the culture, and treated with different reagent concentrations, and pictures were taken of the cells with a microscope (Leica) after 3 or 6 days of culture.

2-3. instruments and reagents used

(ii) ELISA reader Infinite M200(Tecan, Austria), MTT Thiazolidinetetrazole bromide (M5655, Sigma-Aldrich), Onychium spatula (NUNC), Leica microscope

2-4. reagent information

The onion extract powder used was the powder prepared by the above evaluation of the anti-light irradiation skin aging effect, and dimethyl sulfoxide (DMSO) was used as a solvent.

The spiraea glycoside is purchased from the manufacturer (EXTRACSYNTHESE), and the vehicle is dimethyl sulfoxide (DMSO).

2-5. test results

2-5-1. cytotoxicity Assay (MTT Assay)

Toxicity test results of the onion extract powder and meadowfoam are shown in table 4 and fig. 4.

[ TABLE 4 ]

Fibroblast cell

As shown in Table 4 and FIG. 4, since toxicity was not detected in all the concentrations of the onion extract powder and the spiraea glycosides, migration tests were performed using the onion extract powder at different concentrations, such as 50, 100, 250, and 500. mu.g/ml, and the contents of the spiraea glycosides as the index substances, such as 0.15, 0.5, 0.6, 1.5, and 3. mu.g/ml, respectively.

2-5-2. cell migration assay

Wound healing tests were conducted to observe the cell regeneration promoting effects of the onion extract powder and the marker substance spiraein, and the results thereof are sequentially shown in fig. 5 to 15 at different concentrations and on

days

1, 2, 3, 4, 7, and the like.

Wherein, fig. 5 is a comparison result, fig. 6 is a FBS (dental bone serum) 2% treatment result, fig. 7 is onion extract powder 25 μ g/ml, fig. 8 is onion extract powder 50 μ g/ml, fig. 9 is onion extract powder 100 μ g/ml, fig. 10 is onion extract powder 250 μ g/ml, fig. 11 is spiraeoside 0.15 μ g/ml, fig. 12 is spiraeoside 0.3 μ g/ml, fig. 13 is spiraeoside 0.6 μ g/ml, fig. 14 is spiraeoside 1.5 μ g/ml, fig. 15 is spiraeoside 3 μ g/ml treatment result.

As shown in fig. 5 to 15, the experimental results showed that the meadowfoam showed concentration-dependent migration efficacy, and all concentrations of the onion extract powder showed migration efficacy, and especially cell regeneration promoting efficacy similar to that of the positive control FBS at 2% was observed at the concentrations of 50, 100 μ g/ml.

From this, it was determined that the onion extract powder and spiraea glycosides act to promote cell regeneration in wounds.

Although the present invention has been described with respect to the preferred embodiments, various modifications and changes can be made without departing from the spirit of the invention, and the appended claims are intended to cover all such modifications and changes as fall within the true scope of the invention.

Claims

1. A method for preparing a composition for preventing skin damage caused by ultraviolet rays and promoting skin regeneration, the composition comprising an onion extract prepared by the steps of:

stage 1 of immersing dried onion in ethanol to obtain onion extract;

stage 2 of filtering the onion extract;

stage 3 of concentrating the filtered onion extract;

dissolving the solid content in the concentrated solution to 5-20 wt% in the 4 th stage; and

a 5 th stage of removing the nonpolar component in the solution after extracting the solution with hexane and ethyl acetate in sequence,

the content of the spiraea glycoside of the onion extract is 10.0 wt% -40.0 wt%, and the peculiar smell of the onion is removed.

2. The method for producing a composition for preventing skin damage caused by ultraviolet rays and promoting skin regeneration according to claim 1, wherein,

after the above-mentioned phase 5 has been completed,

further comprises a 6 th stage of concentrating the extract under reduced pressure to obtain onion extract powder.

3. The method for producing a composition for preventing skin damage caused by ultraviolet rays and promoting skin regeneration according to claim 1, wherein,

in the above stage 1 onion extract obtaining process,

mixing dried onions: soaking dry onion in 95% ethanol at normal temperature for 1-5 days, and extracting.

4. The method for producing a composition for preventing skin damage caused by ultraviolet rays and promoting skin regeneration according to claim 1, wherein,

in the above-mentioned filtration process of the 2 nd stage,

extracting with a filter having a mesh size of 0.1-10 μm.

5. The method for producing a composition for preventing skin damage caused by ultraviolet rays and promoting skin regeneration according to claim 1, wherein,

in the above-mentioned concentration process in the 3 rd stage,

concentrating the onion extract at 40-70 deg.C.

6. The method for producing a composition for preventing skin damage caused by ultraviolet rays and promoting skin regeneration according to claim 2,

in the above-mentioned phase 6, the phase,

concentrating the extract at 40-100 deg.C under reduced pressure with 40-550 Torr to obtain onion extract powder.

7. A cosmetic comprising a composition for preventing skin damage caused by ultraviolet rays and promoting skin regeneration, which comprises an onion extract produced by the production method of any one of claims 1 to 6.