KR20110123934A - Method of preparing extracts of wild ginseng stem cell(callus) and adventitious roots and cosmetical compositions for the whitening, uv block and anti-oxidant comprising the same - Google Patents

Abstract

PURPOSE: A composition containing wild ginseng stem cells(callus) and adventitious root extract is provided to ensure skin whitening and antioxidaiton and to protect skin from UV ray. CONSTITUTION: A composition for skin whitening and antioxidation contains wild ginseng stem cells(callus) and adventitious root extract. A method for preparing the extract comprises: a step of producing a large amount of wild ginseng stem cells(callus) and adventitious roots using a bioreactor; a step of isolating extract with skin whitening and antioxidation effect from the stem cells and adventitious roots.

Description

Method of preparing extracts of wild ginseng stem cell (callus) and adventitious roots and cosmetical compositions for the whitening, UV block and anti-oxidant comprising the same}

The present invention relates to a composition effective in whitening, sun protection and antioxidant containing wild ginseng stem cells (calus) and wild ginseng root muscle extract as an active ingredient.

The present invention relates to a composition that is effective in whitening, sun protection and antioxidant, including wild ginseng stem cells (calus) and wild ginseng root muscle extract. More specifically, wild ginseng callus and wild ginseng root muscle induced by tissue culture were inoculated into the medium, cultured and harvested in a bioreactor, and extracted with 50-70% fermentation alcohol to extract ginseng stem cells (calus) and wild ginseng root muscle extract. It relates to a whitening, sunscreen and antioxidant composition containing as a component.

Although wild ginseng has been treated as a panacea for a long time, its efficacy has not been well analyzed scientifically, and there are few scientific evidences because there is little research on the functionalities except basic ingredient comparison.

Plant tissue culture technology is a method of forming a plant in the form of cells or tissues or regenerating the plant by using the unique totipotency of the plant, and nutrient, growth hormone, temperature, etc. By the control of, various experiments that are suitable for the purpose of the experimenter are possible. Mass production techniques are required to produce useful materials using this technology, and bioreactors are used here.

Functional cosmetics, which had already been in high demand in developed countries since the late 1980s, is showing rapid growth in Korea for several years. As income increased, living standards improved and more investments were made in cosmetics to enhance one's own image.As the age-old population increased, the need for anti-aging related cosmetics and white face cosmetics Interest and demand for sunscreen have increased due to increased outdoor activities due to the five-day work schedule. As a result, functional cosmetics are diversified and developed more rapidly, showing high growth rates.

Skin is the body's primary protective shield that protects organs in the body from stimuli from the external environment and plays an important role in maintaining homeostasis such as temperature control. However, due to external stimulation such as stimulation of physical and chemical environment, stress and malnutrition, and aging due to life phenomena, the normal function of the skin is lowered and the phenomenon of elasticity loss, keratinization and wrinkle formation is promoted. In order to prevent this phenomenon and maintain a healthier and more elastic skin, by using cosmetics enhanced with conventional bioactive ingredients obtained from various plants and animals and microorganisms, the skin's inherent functions are maintained and skin cells are effectively suppressed by activating skin cells. Efforts have been made to do this.

However, existing cosmetic ingredients have various problems, such as their inefficiency or cause skin side effects, and research on various ingredients to solve them is being actively conducted. It's going on.

Ginsenosides, which are the main functional ingredient of, are already known as functional ingredients in various parts such as antioxidant activity, wound healing, cancer cell metastasis, liver protection, cognitive function and anti-inflammatory action. Ginsenoside, the main pharmacological component, is an ether-bonded compound of sugars such as glucose, rhamnose, arabinose and xylose to the protoparnaxadiol, protoparanaxadiol, and the dimaran-based triterpenoids. More than 30 derivatives have been identified. In addition, ginsenoside derivatives of ginseng and red ginseng have been found to be different depending on the type of sugar, the number and location of the sugars bound to aglycone, and the pharmacological effects of the ginseng are different.

The present inventors searched for the effects on various functionalities related to cosmetics using wild ginseng stem cells (calus) and wild ginseng root muscle, and found that they showed excellent effects on whitening, sun protection and antioxidant activity.

The present invention is to induce wild ginseng stem cells (calus) and wild ginseng root muscle from wild ginseng using tissue culture technology, and mass production using a bioreactor to produce a concentrated solution through the extraction process.

The present invention is to provide a functional cosmetic raw material having a whitening, UV protection and antioxidant effect containing the composition as an active ingredient.

In order to achieve the above object, the present inventors establish conditions for inducing stem cells (calus) and root muscles from wild ginseng under aseptic environment, and after harvesting by proliferating each tissue in a bioreactor under the established conditions, Using ginseng stem cells (calus) and wild ginseng root muscle extract concentrate having a whitening, UV protection and antioxidant effect by using to complete the present invention.

Accordingly, an object of the present invention is to induce wild ginseng stem cells (calus) and wild ginseng root muscle using plant tissue culture technology and culture using a bioreactor.

In addition, an object of the present invention is to produce a concentrated solution after extracting wild ginseng stem cells (callus) and wild ginseng root muscle which are effective in whitening, UV protection and antioxidant.

Plant tissue culture technology and bioreactor can mass-produce wild ginseng stem cells (calus) and wild ginseng root muscle which are effective in whitening, UV protection and antioxidant. This solves the problem of obtaining the most problematic sample in wild ginseng research, and it can be a good stepping stone for the research of wild ginseng, which lacks scientific evidence despite various outstanding effects.As a sterile production process, no residual pesticides and heavy metals are detected. The impact of well-being can meet the needs of consumers looking for clean ginseng. In addition, by showing the functional effects related to cosmetics, it can be used as an excellent herbal material to challenge the global cosmetics market of $ 344.9 billion as of 2010.

1 is a cell picture and graph confirming the melanin inhibition rate by treating the ginseng stem cell (callus) extract of the present invention to B16 melanoma cells by concentration.
Figure 2 is a cell picture and a graph confirming the melanin inhibition rate by treating the mountain ginseng root muscle extract of the present invention to B16 melanoma cells by concentration.
3 is a gel (gel) and cell staining pictures and graphs confirming the inhibition of tyrosinase activity, a melanin production promoting enzyme, by treating the ginseng stem cell (callus) extract of the present invention to B16 melanoma cells by concentration.
4 is a gel (gel) and cell staining photographs and graphs confirming the inhibition of tyrosinase activity, a melanin production promoting enzyme, by treating the wild ginseng root muscle extract of the present invention in B16 melanoma cells at different concentrations.
Figure 5 is a photograph and graph confirming the cell survival rate by UV irradiation after the treatment of wild ginseng stem cell (Calus) extract of the present invention to human normal fibroblasts (CCD-986sk cell) by concentration.
Figure 6 is a photograph and a graph confirming the cell survival rate by ultraviolet irradiation after treatment of wild ginseng root muscle extract of the present invention to human normal fibroblasts (CCD-986sk cell) by concentration.
7 is treated with H ₂ O ₂ (active oxygen) after treatment of the wild ginseng stem cell (callus) extract of the present invention to the normal fibroblasts (CCD-986sk cell) of each concentration to protect the cells from the effects of free radicals Is a graph confirming the cell viability.
8 is treated with H ₂ O ₂ (active oxygen) after treatment of wild ginseng root muscle extract of the present invention to the normal fibroblasts (CCD-986sk cell) of the present invention to the cell survival rate from the effect of free radicals Checked graph.
9 is a primary protective membrane of the active oxygen in the cells after treatment with the concentration of wild ginseng stem cells (callus) of the present invention to the normal fibroblasts (CCD-986sk cell) of the present invention and H ₂ O ₂ (active oxygen) This is a graph confirming the activity of the phosphorus antioxidant enzyme SOD (Superoxide dismutase).
10 is an antioxidant enzyme SOD that is the primary protective film of active oxygen in cells after treatment with the concentration of wild ginseng root muscle of the present invention in human normal fibroblasts (CCD-986sk cell) at different concentrations and H ₂ O ₂ (active oxygen). This graph shows the activity of (Superoxide dismutase).
Figure 11 is treated with the concentration of wild ginseng stem cells (callus) of the present invention to the normal fibroblasts (CCD-986sk cell) of the present invention and treated with H ₂ O ₂ (active oxygen) after the intracellular antioxidant enzyme GSH (Glutathione) ) Is a graph confirming the activity of.
FIG. 12 shows the activity of GSH (Glutathione), an intracellular antioxidant enzyme, after treatment of wild ginseng sarcophagus extract of the present invention to human normal fibroblasts (CCD-986sk cell) by concentration and H ₂ O ₂ (active oxygen). Checked graph.

Composition of the Invention

According to the present invention,

(Iii) aseptically inducing wild ginseng stem cells (calus) and wild ginseng root muscle from wild ginseng and mass production using a bioreactor;

(Ii) using a mass-produced wild ginseng stem cells (calus) and wild ginseng root muscle to provide a method for producing an extract that is effective in whitening, sun protection and antioxidant.

Hereinafter, the method of the present invention will be described in detail step by step.

Ⅰ. Inducing wild ginseng stem cells (calus) and wild ginseng root muscle aseptically from wild ginseng and mass production using a bioreactor;

Sterilize sterilized ginseng of the above step (ⅰ), cut into 1-2 mm thickness, peel off the endothelium, separate the cambium from the somewhat woody internal tissue, and then remove 2,4-dichlorophenoxy acetic acid from 1.0 to 10.0. Stem cells (callus) are induced by inoculating modified Schenk & Hildebrandt medium with 2,4-dichlorophenoxy acetic acid in an amount of mg / L, preferably in an amount of 1.0-10.0 mg / L. The induced stem cells (calus) were grown in modified Schenk & Hildebrandt medium to which 1.0 mg / L of 2,4-dichlorophenoxy acetic acid was added, and indolebutyl acetic acid was 5 to 10 while passaged at 2 week intervals. The amount of mg / L is transferred to modified Schenk & Hildebrandt medium, which is preferably added in an amount of 5 mg / L, to form a root muscle. The cultured wild ginseng stem cells (calus) were suspended in the same medium, and random roots were randomly cut to a size of 0.5 to 1 cm and inoculated into an air flotation bioreactor. At this time, the culture medium contains pH 5.8 ~ 6.0, sugar concentration 3 ~ 7% and vitamin in modified Schenk & Hildebrandt medium and growth regulator was treated at the same concentration as that added at the time of induction and air injection amount was 300 ~ 700 cc / Incubate at 18-24 degreeC temperature conditions as min.

Ⅱ. Preparing extracts that are effective in whitening, sun protection and antioxidant using mass-produced wild ginseng stem cells (calus) and wild ginseng root muscles;

The wild ginseng stem cells (calus) and wild ginseng root muscle obtained in the above step (ⅰ) are washed and dried by lyophilization and hot air drying, respectively, and the volume of water, fermentation alcohol or their equivalent to 5 to 10 times the sample, respectively. Mixed solvent, preferably 50-70% fermented alcohol extract using extraction method such as reflux cooling extraction or hot water extraction for 8 to 24 hours at 60 ~ 80 ℃, then filtered and concentrated under reduced pressure to extract wild ginseng stem cells (Calus) ) And wild ginseng root extract.

Example  1: wild ginseng stem cell ( Callus ) And Mountain ginseng  Preparation of Extract

1-1. Induction of Wild Ginseng Stem Cells (Calus) and Wild Ginseng Muscles by Tissue Culture

After sterilizing the roots of wild ginseng, cutting it to a thickness of 1 to 2 mm, peeling off the endothelium, separating the cambium from the somewhat woody internal tissue, and then removing 1.0 to 10.0 mg / L of 2,4-dichlorophenoxy acetic acid. Stem cells (callus) were induced by applying to modified Schenk & Hildebrandt medium added in the amount of. Induced stem cells (calus) were grown in modified Schenk & Hildebrandt medium supplemented with 2,4-dichlorophenoxy acetic acid in an amount of 1.0 mg / L, and 5 mg of indolebutyl acetic acid was passaged every two weeks. Nerve root was formed by transferring to modified Schenk & Hildebrandt medium added in the amount of / L.

1-2. Mass production of wild ginseng root muscle containing a lot of induced ginseng stem cells (calus) and saponin

The cultured wild ginseng stem cells (calus) were suspended in the same medium, and random roots were randomly cut to a size of 0.5 to 1 cm and inoculated into an air flotation bioreactor. At this time, the culture medium contained pH 5.8, sugar concentration 3% and vitamin in modified Schenk & Hildebrandt medium. Growth regulator was treated at the same concentration as that added at the induction. Incubate at a temperature condition of ℃.

1-3. Preparation of Mass-produced Wild Ginseng Stem Cells (Calus) and Wild Ginseng Parotid Root Extracts

The wild ginseng stem cells (calus) and wild ginseng root muscle are washed and dried by lyophilization and hot air drying, respectively, and 50 times or 70 times fermented alcohol corresponding to 8 or 10 times the sample volume, respectively, three times at 80 ° C. for 8 hours. Extracted by extraction method such as reflux cooling extraction or hot water extraction for 24 hours, and then filtered and concentrated under reduced pressure to obtain wild ginseng stem cells (calus) and wild ginseng root muscle extract.

Example  2: wild ginseng stem cell ( Callus ) And Mountain ginseng  Measurement of Cosmetic Functional Effects of Extracts

The wild ginseng stem cells (callus) and wild ginseng root muscle extract obtained in 1-3 of Example 1 were used.

2-1. Whitening effect

Melanin production inhibition

The wild ginseng stem cells (calus) obtained from Example 1 and the wild ginseng root muscle extract showed whitening effects using B16 melanoma cells, which are cultured cells.

The melanin glass and the amount of production were measured by Hosoi et al. B16 melanoma cells were inoculated at an appropriate cell concentration and incubated for 24 hours. After treatment with α-MSH (melanocyte stimulating hormone), wild ginseng stem cell (calus) extract, and wild ginseng root muscle extract, the cells were incubated for 3 days and containing 10% dimethyl sulfoxide. 100 μl of 1 N NaOH was added, followed by reaction at 75 ° C. for 1 hour, and the absorbance was measured at 420 nm with an ELISA reader to determine the amount of melanin.

As a result, it was confirmed that melanin production decreased as the concentration of both ginseng stem cell (callus) extract and wild ginseng root muscle extract increased. In particular, the extract of wild ginseng stem cells (calus) has little effect on the toxicity of the cells and suppressed the production of melanin by more than 40%. The results of the melanin production inhibition according to each extract is shown in Figure 1 (A, B), Table 1, Figure 2 (A, B) and Table 2.

① Wild Ginseng Stem Cell (Callis) Extract

TABLE 1

② Sansambu root muscle extract

TABLE 2

Tyrosinase Active inhibition

B16 melanoma cells were inoculated into 96 well plates and inoculated at an appropriate cell concentration for 24 hours. After treatment with α-MSH (melanocyte stimlating hormone), wild ginseng stem cell (calus) extract and wild ginseng root muscle extract, they were incubated for 3 days. After lysis by adding lysis buffer, the supernatant was quantified by centrifugation for 30 minutes. After quantitative protein electrophoresis of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the gel was transferred to 5 mM L-DOPA (L-3,4-dihydroxyphenylalanine) solution and stained for 6 hours at 37 ° C under dark conditions. Tyrosinase activity was analyzed by a -3000 image analyzer.

In addition, 10 mM L-DOPA (L-3,4-dihydroxyphenylalanine) and PBS (phosphate buffered saline) were added to 100 μl of the quantified supernatant, and reacted at 35 ° C. for 1 hour, and then the absorbance was measured at 475 nm using an ELISA reader. Tyrosinase activity was confirmed.

As a result, the production of DOPA, which is a result of tyrosinase activity, decreased as the concentration of both ginseng stem cell (calus) extract and wild ginseng root muscle extract increased. About 27% of wild ginseng stem cell (calus) extracts and wild ginseng root muscle extracts inhibited tyrosinase activity by more than about 55%. Results of melanin production inhibition according to each extract are shown in Figure 3 (A, B, C), Table 3 and Figure 4 (A, B, C).

 Wild Ginseng Stem Cell Extract

[Table 3]

 Wild ginseng root muscle extract

[Table 4]

2-2. UV protection effect

The effects of wild ginseng stem cells (callus) and wild ginseng root muscle extracts obtained from Example 1 on UV protection and antioxidant activity were identified using CCD-986sk cells (human fibroblast cell line).

Mosmann (1983), Kotnik (1990) and others were used for the protection against ultraviolet rays. CCD-986sk cells were inoculated into 96 well plates at an appropriate cell concentration and incubated for 24 hours. After treatment with wild ginseng stem cell (calus) extract and wild ginseng root extract, they were treated under UV conditions for 10 minutes and incubated for 20 hours, followed by MTT (3- (4,5-dimethylthiazol-2yl) -2,5-diphenyl-2H-tetrazolium bromide) was added and incubated for 4 hours at 37 ° C under dark conditions. After incubation, dimethyl sulfoxide was added to each well, followed by reaction at 37 ° C. for 1 hour, and the cell viability was confirmed by measuring absorbance at 540 nm with an ELISA reader.

As a result, when the wild ginseng stem cell (callus) extract and wild ginseng root muscle extract were treated, it was confirmed that the cell protective effect against ultraviolet rays was increased by 22-32% at a certain concentration. The results of the protective effect against ultraviolet rays according to each extract are shown in Figure 5 (A, B), Table 5, Figure 6 (A, B) and Table 6.

 Wild Ginseng Stem Cell Extract

TABLE 5

 Wild ginseng root muscle extract

TABLE 6

2-3. Antioxidative effect

H ₂ O ₂ Protection against

Mosmann (1983), Kotnik (1990) and others were used to protect the hydrogen peroxide. CCD-986sk cells were inoculated in 96 well plates at the appropriate cell concentration and incubated for 24 hours. After treatment with wild ginseng stem cell (calus) extract, wild ginseng root muscle extract and H ₂ O ₂ , the cells were incubated for 20 hours and then MTT (3- (4 , 5-dimethylthiazol-2yl) -2,5-diphenyl-2H-tetrazolium bromide) was added and incubated for 4 hours at 37 ° C under dark conditions. After incubation, dimethyl sulfoxide was added to each well, followed by reaction at 37 ° C. for 1 hour, and the cell viability was confirmed by measuring absorbance at 540 nm with an ELISA reader.

As a result, it was confirmed that the cell protective effect against free radicals in both wild ginseng stem cell (calus) extract and wild ginseng root muscle extract were increased. The protective effect was increased by more than 13 ~ 17% when treated with specific concentration than without extract. The results of the protective effect on the active oxygen according to each extract is shown in Figure 7, 8 and Table 7, 8.

 Wild Ginseng Stem Cell Extract

TABLE 7

 Wild ginseng root muscle extract

[Table 8]

Antioxidant activity ( SOD activation)

Confirmation of SOD (Superoxide dismutase) activity, which is the primary protective film of reactive oxygen,

It confirmed through.

Inoculate CCD-986sk cells in 96-well plates at the appropriate cell concentration and incubate for 24 hours. After treatment with wild ginseng stem cell (calus) extract, wild ginseng root muscle extract and H ₂ O ₂ , incubate for 3 hours, and add lysis buffer The supernatant was quantified by centrifugation for two times for 40 minutes. The quantitated protein was mixed with the nitrate-producing solution for 20 minutes at 25 ° C, 1.65 mg of sulfanilic acid and 0.5 mg α-naphthyamine were added and reacted at room temperature for 20 minutes. Confirmed.

As a result, it was confirmed that SOD activity was increased in both wild ginseng stem cell (calus) extract and wild ginseng root muscle extract. In particular, wild ginseng root muscle extract showed more than 47% SOD activity when treated at a certain concentration than when not treated. The results of the protective effect on the active oxygen according to each extract is shown in Figures 9, 10 and Tables 9 and 10.

 Wild Ginseng Stem Cell Extract

TABLE 9

 Wild ginseng root muscle extract

TABLE 10

Antioxidant activity ( GSH activation)

Inoculate CCD-986sk cells in 96-well plates at the appropriate cell concentration and incubate for 24 hours. After treatment with wild ginseng stem cell (calus) extract, wild ginseng root muscle extract and H ₂ O ₂ , incubate for 3 hours, and add lysis buffer The supernatant was quantified by centrifugation for two times for 40 minutes. The quantified protein was mixed with GSH (Glutathione) assay buffer and reacted at 20 ° C. for 5 minutes, and immediately after treatment with H ₂ O ₂ , the absorbance was measured at 410 nm with an ELISA reader to confirm GSH activity.

As a result, GSH activity was increased in both wild ginseng stem cell (calus) extract and wild ginseng root muscle extract. In particular, GSH activity was increased by 30% or more when treated at a certain concentration than in the case of wild ginseng root muscle extract. The results of the protective effect on the active oxygen according to each extract is shown in Figures 11 and 12 and Tables 11 and 12.

 Wild Ginseng Stem Cell Extract

TABLE 11

 Wild ginseng root muscle extract

TABLE 12

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

There is no corresponding information.

Claims (2)

Aseptic method for inducing wild ginseng stem cells (calus) and wild ginseng root muscle from wild ginseng and mass production using bioreactor
Method for producing extracts effective in whitening, sun protection and antioxidant using mass-produced wild ginseng stem cells (calus) and wild ginseng root muscle

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