KR20190006126A - High yield ginsenoside biosynthesis inducing composition obtained from cultured ginseng callus cell of Korean and their uses - Google Patents

Abstract

The present invention relates to wild ginseng stem cell culture extract, a method to manufacture the same, a cosmetic composition containing the same, and a pharmaceutical composition including the same. More particularly, the present invention relates to a wild ginseng stem cell culture extract produced by artificially increasing ginsenoside which is a unique pharmacological component expressed in Korean wild ginseng, and to a method to manufacture the same. The method to manufacture the wild ginseng stem cell culture extract comprises the following steps: obtaining wild ginseng callus from MS medium; culturing the wild ginseng callus by cell suspension culture; and performing freeze-drying.

Description

{High yield ginsenoside biosynthesis inducing composition obtained from cultured ginseng callus cell of Korean and their uses}

The present invention relates to a culture extract of wild ginseng stem cells, a method for producing the same, a cosmetic composition containing the same, and a pharmaceutical composition containing the same, and more particularly, to a method for artificially increasing ginsenoside, Cultured ginseng stem cell culture extract and a method for producing the same.

Unlike cultured ginseng, wild ginseng is known to express a unique gene structure and high quality ginsenosides biosynthesized from it. There have been many attempts to utilize wild ginseng as biomaterials through biotechnological means. However, most of them have been in the field of producing ginseng cultivation roots that artificially induce adventitious roots through tissue culture, and in the field of primary foods that are used for the cultivation or drying.

The ginseng cultivar is a delicate thread-like fine roots like ginseng root, which is mass-produced after being introduced through a bioreactor, and once it is differentiated into root tissue. The embryological value of the cultivating muscle itself is not considered to be undifferentiated because it corresponds to the roots of the grown wild ginseng.

On the other hand, when undifferentiated stem cells are induced from wild ginseng tissue and cultured in a solid medium, they develop into cell masses, which are collectively referred to as calli. These undifferentiated cell masses do not differentiate into specific tissues and possess the omnipotence of substance synthesis. If artificial mass expression of the stem cell substance synthesis system, which can be expressed under callus conditions, can be achieved, a novel substance that can not usually be found can be derived.

The present invention aims to produce a new ginseng stem cell culture extract which has not been known by cultured root system induced by ginseng root ginseng but by mass culture of undifferentiated stem cells and promotion of artificial metabolite biosynthesis. Through this, it is possible to overcome the limitation of production process of various ginsenoside compositions from the inherently pharmacologically active substances of wild ginseng, which is not normally found in ginseng or red ginseng and can not be overcome so far.

Accordingly, an object of the present invention is to provide a culture extract of wild ginseng stem cells.

It is still another object of the present invention to provide a method for producing an extract of cultured wild ginseng stem cells.

Another object of the present invention is to provide a cosmetic composition comprising a culture extract of wild ginseng stem cells.

It is still another object of the present invention to provide a pharmaceutical composition comprising a cultured ginseng stem cell culture extract.

The present invention relates to a culture extract of wild ginseng stem cells, a method for producing the same, a cosmetic composition containing the same, and a pharmaceutical composition containing the same, and more particularly, to a method for artificially increasing ginsenoside, Cultured ginseng stem cell culture extract and a method for producing the same.

Hereinafter, the present invention will be described in more detail.

One example of the present invention relates to a method for producing an extract of cultured ginseng stem cell.

The process may comprise the following steps:

Obtaining a wild ginseng callus in which a Korean wild ginseng slice is tissue-cultured in an MS (Murashige and Skoog) medium supplemented with sucrose, 2,4-dichlorophenoxy acetic acid and gelite;

Culturing suspension cells in which MS ginseng callus is treated with methyl jasmonate or methyl jasmoic acid in an MS medium supplemented with sucrose and 2,4-dichlorophenoxyacetic acid to culture floating cells; And

Removing the culture medium, mixing the distilled water, and freeze-drying using a freeze dryer.

The step of obtaining the ginseng callus may be performed by tissue culture in MS (Murashige and Skoog) medium supplemented with 3% sucrose, 1 mg / L 2,4-dichlorophenoxyacetic acid and 2.4 g / L gelite.

The methyl jasmonate may be treated with 200 uM.

Wild ginseng is a perennial herb that is traditionally very expensive, due to its superior pharmacological properties compared to ginseng. It is known that the major constituent of pharmacological components of wild ginseng is a ginsenoside, which is unique to ginseng or its red ginseng.

Wild ginseng is a perennial natural herb that is considered to be a mysterious medicine from ancient times to the present. We intensively developed the naturally ginseng ginsenoside as a unique ginsenoside composition of wild ginseng cells through cell culture using a bioreactor and tried to develop this material for commercialization.

Wild ginseng stem cells are derived from the vigorous dividing growth splitting tissue into tissue culture, and the formed tissue is callus type undifferentiated cell mass.

Therefore, the present inventors obtained callus derived from the ginseng derived from Korean ginseng and proliferated through suspension cell cultures, and then treated with methyl jasmonate or methyl jasmonate to increase the synthesis of secondary metabolites during cell proliferation and growth And cells were obtained.

The obtained cells were assayed for ginsenoside and saponin content by mass spectrometry or absorbance method.

As a result, total ginsenosides induced by methyl jasmonate or methyl jasmonic acid treatment were 85% and 22% increased in wild ginseng callus and conditioned medium (CM), respectively, And the side change amount was remarkably increased and ascended by 286%.

The amount of ginsenoside composition obtained was 94.1% in the control group and 69.8% in the treatment group MeJA2, which was composed of Rb1, Rg1 and Rg3. Thus, a considerable amount of ginsenoside downstream metabolism proceeded to the ginsenoside downstream metabolism .

The ginseng stem cell culture extract (named RFG17) of the present invention contains Rd, F2 and Gp17, and the total ginsenoside content is increased by more than 9 times (2.5% to 23.3%) and the molecular constitution Rd, F2, Gp17 is about 7: 1: 3.

The cultured ginseng stem cell culture extract shows major activity for skin cell anti-aging.

Culturing of wild ginseng stem cells can be carried out by inducing the tissue-cultured callus to micronized cell bodies by micrometry and inoculating the cells into a stereogenic apparatus such as a bioreactor or a Rotary Cell Culture System (RCCS) .

The suspension cell culture may be cultured for 10 to 30 days, 10 to 25 days, 10 to 20 days, 10 to 15 days, 12 to 30 days, 12 to 25 days, 12 to 20 days, 12 to 15 days, 14 to 30 days, 14 to 25 days, 14 to 20 days, or 14 to 15 days, for example, 14 days.

In order to artificially amplify the induction of ginsenoside biosynthesis according to the proliferation of wild ginseng stem cells, methyl esters of methyl esters or methyl jasmonate, which is known to exhibit the effect of promoting secondary metabolites in plant tissue culture, 8 days, 3 to 7 days, 3 to 6 days or 3 to 5 days, for example, for 3 to 5 days.

Methyl jasmonate or methyl jasmonate is added to the proliferated cultured cell culture at a concentration of 10 to 1000 uM, 10 to 900 uM, 10 to 800 uM, 10 to 700 uM, 10 to 600 uM, 10 to 500 uM, 10 to 400 uM 50 to 1000 uM, 50 to 900 uM, 50 to 800 uM, 50 to 700 uM, 50 to 600 uM, 50 to 500 uM, 50 to 400 uM, 50 to 300 uM, 100 to 1000 uM , 100 to 900 uM, 100 to 800 uM, 100 to 700 uM, 100 to 600 uM, 100 to 500 uM, 100 to 400 uM or 100 to 300 uM, Lt; RTI ID = 0.0 > uM. ≪ / RTI > Within the above range, treatment promotes the synthesis of cellular metabolites.

The wild ginseng stem cells and their cell culture (CM) may be separated by filtration through a final 300 micron mesh, but are not limited thereto.

The ginseng cells obtained from the view flow cell culture are preserved by freeze drying, and the filtered cell culture solution is stored after high pressure steam sterilization.

The ginsenoside cell culture extract may have a ginsenoside concentration of 6 mg / g or more by LC / Q-Orbitrap assay.

The total concentration of saponin in the cultured ginseng stem cell culture extract may be 1% to 3% by a sulfuric acid-vanillin chromogenic assay using an absorbance meter.

The cultured ginseng stem cell culture extract may have a weight ratio of biosynthesis of Rg1, Rb1, and Rg3 of 60 to 80% of total ginsenoside weight.

The ginseng stem cell culture extract may have a total ginsenoside content increased by 200 to 400% as compared to the non-treated group of methyl jasmonate.

The ginseng stem cell culture extract has a total ginsenoside content of 5.0 to 8.0 mg / g, 5.0 to 7.9 mg / g, 5.0 to 7.8 mg / g, 5.1 to 8.0 mg / g, 5.1 to 7.9 mg / 7.8 mg / g, for example 5.14 to 7.71 mg / g.

Another example of the present invention relates to a ginseng stem cell culture extract.

The ginseng stem cell culture extract may have a total saponin content of 20.5 mg / g or more.

In addition, the ginseng stem cell culture extract may have a total ginsenoside content of 6.0 mg / g or more.

The content of Rd, F2 and Gp17 contained in the cultured ginseng stem cell culture extract may be 10 to 30% of the total ginsenoside content.

The content ratio of Rd: Gp17: F2 contained in the cultured ginseng stem cell culture extract may be 7: 3: 1.

Another example of the present invention relates to a cosmetic composition for improving skin condition comprising the cultured ginseng stem cell culture extract.

The improvement of the skin condition is wrinkle improvement, skin elasticity improvement, skin aging prevention, skin moisturizing improvement, acne improvement or whitening.

The cosmetic composition may be a cosmetic composition comprising a cosmetically effective amount of the ginseng stem cell culture extract and / or a cosmetically acceptable carrier.

The term " cosmetically effective amount " as used herein means an amount sufficient to achieve the skin-improving effect of the composition of the present invention described above.

The cosmetic composition may be in any form conventionally prepared in the art and may be in the form of, for example, a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant- , A powder foundation, an emulsion foundation, a wax foundation, and a spray, but is not limited thereto. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

When the formulation of the cosmetic of the present invention is a paste, a cream or a gel, an animal oil, a vegetable oil, a wax, a paraffin, a starch, a tracer, a cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, Can be used.

When the formulation of the cosmetic of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in the case of a spray, additionally a chlorofluorohydrocarbon , Propane / butane or dimethyl ether.

When the formulation of the cosmetic of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid esters of sorbitan.

When the formulation of the cosmetic composition of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspension such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the cosmetic of the present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, Fatty acid amide ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester.

The components contained in the cosmetic composition of the present invention include components commonly used in cosmetic compositions, in addition to the ginseng stem cell culture extract and carrier component as an active ingredient. Examples thereof include antioxidants, stabilizers, solubilizers, vitamins, Pigments, and flavoring agents.

Another example of the present invention relates to a pharmaceutical composition for preventing skin aging comprising a cultured ginseng stem cell culture extract.

In addition, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carriers are those conventionally used in the formulation and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, But are not limited to, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may further include, but is not limited to, lubricants, wetting agents, sweetening agents, flavoring agents, emulsifying agents, suspending agents, preservatives, etc. in addition to the above components.

The pharmaceutical composition may be administered orally or parenterally, preferably parenterally. In the case of parenteral administration, the pharmaceutical composition may be administered by intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, topical administration, transdermal administration or the like , But is not limited thereto.

The dosage of the pharmaceutical composition may be from 0.0001 to 1000 ug (microgram, 0.001 to 1000 ug, 0.01 to 1000 ug, 0.1 to 1000 ug, or 1.0 to 1000 ug per day, but is not limited to, May be variously prescribed by factors such as the method of administration, the mode of administration, the age, body weight, sex, pathological condition, food, administration time, route of administration, excretion rate and responsiveness of the patient.

The pharmaceutical composition may be prepared in the form of a unit dose by formulating it with a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those having ordinary skill in the art to which the present invention belongs. Into a multi-dose container.

The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing agents and / or stabilizers.

The present invention aims to produce a culture extract of wild ginseng stem cells with a novel ginsenoside ratio which is not known through the process of mass culture of undifferentiated stem cells and promotion of an artificial metabolite biosynthesis, Through this, it is possible to overcome the limitation of production process of various ginsenoside compositions from the inherently pharmacologically active substances of wild ginseng, which is not normally found in ginseng or red ginseng and can not be overcome so far.

FIG. 1 is a view showing a process of producing a wild ginseng callus for cell culture by Korean wild ginseng tissue culture according to an embodiment of the present invention.
FIG. 2 is a graph showing the production of amplified ginsenoside-expressing Korean wild ginseng cells by suspension cell culture according to an embodiment of the present invention.
FIG. 3A is a graph showing the total saponin content according to one embodiment of the present invention. FIG.
FIG. 3B is a graph showing the total saponin content according to an embodiment of the present invention. FIG.
Figure 4 is a graph showing total ginsenoside content according to one embodiment of the present invention.
Figure 5 is a graph showing total ginsenoside content comparisons of comparative examples according to one embodiment of the present invention.
Figure 6 is a graph showing total ginsenoside content comparisons of an example according to one embodiment of the present invention.
FIG. 7 is a graph showing the content of the intelligent lipid component in the ginsenoside contents in the comparative examples and the examples according to an embodiment of the present invention.
FIG. 8 is a graph showing the contents of research-specific components in Comparative Examples and Examples according to an embodiment of the present invention.
9 is a graph showing a comparison of contents of Rd, Gp17 and F2 according to an embodiment of the present invention.
10 is a graph showing the composition ratio of Rd, F2, and Gp17 in accordance with an embodiment of the present invention.
11 is a graph showing the compositional composition ratio of Rd, F2, and Gp17 according to an embodiment of the present invention.
12 is a graph showing the compositional composition ratio of Rd, F2, and Gp17 in the embodiment according to one embodiment.
13 is a graph showing the compositional composition ratio of Rd, F2, and Gp17 in the comparative example according to one embodiment.
FIG. 14 is a graph showing the compositional composition ratio of Rd, F2, and Gp17 according to an embodiment of the present invention.
15 is a graph showing the compositional composition ratio of Rd, F2, and Gp17 according to an embodiment of the present invention.
16 is a graph showing antioxidant efficacy (DPPH) of a cosmetic composition according to an embodiment of the present invention
17 is a graph showing the antioxidative activity (SOD-like activity) of the cosmetic composition according to one embodiment of the present invention.
18 is a graph showing the whitening efficacy (tyrosinease inhibition, L-tyrosine) of a cosmetic composition according to an embodiment of the present invention.
19 is a graph showing whitening efficacy (tyrosinease inhibition, L-DOPA) of a cosmetic composition according to an embodiment of the present invention
20 is a graph showing the wrinkle-reducing effect (elastin degrading enzyme inhibition) of the cosmetic composition according to one embodiment of the present invention.
21 is a graph showing the wrinkle-reducing effect (hyaluronic acid degrading enzyme inhibition) of the cosmetic composition according to one embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1. Production of wild ginseng callus

1-1. Induction of wild ginseng callus

Six roots of Deoksu Sanjungsan ginseng, which were inspected by Korean Traditional Simmatic Society, were washed in running water for 24 hours, and then the contaminated source was removed with a soft toothbrush, and then a part of the epidermis was removed with a knife to prepare the deodorant solution to penetrate deeper .

Then, 50 ㎕ of Tween 20 was added to 2% sodium hypochlorite (NaOCl), and the main muscle was sterilized for 20 minutes and the fine muscle was sterilized for 15 minutes, and then washed with sterilized water three times or more, . Then, the epidermis was removed to prevent damage to the cambium layer of the main muscle, and the hair was cut into 0.5 cm ³ size including the cambium layer. The cuticle was cut into 1 cm length and implanted in adventitious root induction medium.

1-2. Induction callus selection

The sections grown in the induction medium were observed daily to check for contamination, and the pincette was carefully sterilized to prevent contamination during passage. Through continuous observation and subculture, growth and clear callus were continuously selected and proliferated. The callus from the section was isolated and subcultured and screened to obtain the best cell cultured ginseng callus.

Specifically, the medium was sterilized in a high-pressure sterilizer at 121 ° C and 1.2 atm with 1 MS (Murashige and Skoog), 3% sucrose, 1 mg / L 2,4-dichlorophenoxyacetic acid and 2.4 g / And 40 ml each were used in petri dishes. The cells were cultured at 23 ± 1 ° C and subcultured at intervals of 2 weeks.

Example 2. Culture of ginseng callus cells and production of ginsenoside

2-1. Culture of wild ginseng callus cells

The selected callus was subcultured on a plate every 2 weeks in 1MS, 3% sucrose, 1 mg / L 2,4-dichlorophenoxyacetic acid and 2.4 g / L gelite medium and cultured at 23 ± 1 ° C. (1 MS, 3% sucrose, 1 mg / L 2,4-dichlorophenoxyacetic acid) except for gelite to scale up with a liquid suspension culture. 100 ml was dispensed into a 300 ml flask and sterilized for 20 minutes at 121 ° C and 1.2 atm. After sterilization, the ginseng callus was inoculated at an inoculum density of 4 to 6% and harvested by incubation in a shaker at 23 ± 1 ° C for 2 weeks.

Then, the callus propagated through liquid suspension culture was diluted with 7 L of liquid medium (1 MS, 3% sucrose, 1 mg / L 2,4-dichlorophenoxyacetic acid) except gelite to scale up After sterilization in a high-pressure sterilizer at 121 ° C and 1.2 atm for 60 minutes, the ginseng callus was inoculated into a bioreactor or RCCS (Rotary Cell Culture System) at an inoculum density of 4 to 6% and harvested by incubation at 23 ± 1 ° C. for 2 weeks . In order to increase the biosynthesis of the material, the elicitor treatment was performed for 10 days after the cultivation.

After the bioreactor was cultured, the culture medium was sampled at a harvest time of 300 .mu.M. The fresh weight was measured, and the medium was removed from the flowing water. The ginseng callus was mixed with a little water, frozen in a cryogenic freezer, Respectively. The dry weight of the final product was measured and sealed and stored in a dark room.

2-2. Cell culture production

For the production of cell culture, the cell culture solution that had been filtered through the cultured cells was finely ground in a mixer and sterilized for 1 hour in a high-pressure sterilizer.

2-2-1. Production of experimental group 1 (MeJA)

7 L of a liquid medium (1 MS, 3% sucrose, 1 mg / L 2,4-dichlorophenoxyacetic acid) except gelite was prepared and sterilized in a high-pressure sterilizer at 121 캜 and 1.2 atm for 60 minutes to obtain an inoculum density of 4 to 6% The ginseng callus was inoculated into a bioreactor or RCCS (Rotary Cell Culture System) and cultured for 2 weeks at 23 ± 1 ℃.

On the 10th day of culture, 200 μM of methyl jasmonate was added to enhance the secondary metabolite biosynthesis, and the culture was collected on the 14th day of cultivation. At that time, the culture medium was collected in a 300-μm sieve at the time of harvesting, And then freeze-dried for 1 week in a cryogenic freezer mixed with a little water.

2-2-2. Production of experimental group 2 (MeJA2)

7 L of a liquid medium (1 MS, 3% sucrose, 1 mg / L 2,4-dichlorophenoxyacetic acid) except gelite was prepared and sterilized in a high-pressure sterilizer at 121 캜 and 1.2 atm for 60 minutes, The ginseng callus was inoculated into a bioreactor or RCCS (Rotary Cell Culture System) and cultured for 2 weeks at 23 ± 1 ℃.

On the 10th day of cultivation, 200 μM of methyl jasmonate was treated to enhance the production of the substance, and after the culture was harvested on the 14th day of culturing, the medium was completely filtered with 300 μm sieve, and CM2 (conditioned media) , Followed by mixing with a little water, storing in a cryocooler, freezing and freezing for one week.

CM2: Mixed ginseng callus with CM (Conditioned media) at 1: 1, and finely ground in a blender, and then sifted through the sieve in the order of 300 μm, 150 μm, and 90 μm.

2-2-3. Control (control) production

7 L of a liquid medium (1 MS, 3% sucrose, 1 mg / L 2,4-dichlorophenoxyacetic acid) except gelite was prepared and sterilized in a high-pressure sterilizer at 121 캜 and 1.2 atm for 60 minutes to obtain an inoculum density of 4 to 6% The ginseng callus was inoculated into a bioreactor or RCCS (Rotary Cell Culture System) and cultured at 23 ± 1 ℃ for 2 weeks.

 On the 14th day of cultivation, the medium was removed to 300 체 sieve at the time of harvest, and then the medium was removed from the flowing water. Then, the ginseng callus was mixed with a little water and stored in a cryocooler, frozen, and lyophilized for one week.

Example 3. Analysis of cell culture

3-1. Ginsenoside content analysis

The total ginsenoside content of wild ginseng cells and cell culture obtained through suspension cell culture was analyzed, and the results are shown in FIGS. 3A and 3B.

Percentage vs. Control (%) Stdev. MeJA 198.554 0.639 MeJA2 185.311 13.115 Control group 100,000 5.087

Percentage vs. Control (%) Stdev. MeJA2 122.177 2.102 Control group 100,000 2.210

As shown in FIGS. 3A and 3B, the total saponin content of the cultured ginseng stem cells was> 22% higher than that of the control, and the total saponin content of the ginseng stem cells was> 185% higher than that of the control.

3-2. Saponin content analysis

The total saponin content of wild ginseng cells and cell culture obtained through floating cell cultures was analyzed and the results are shown in FIG. 4 and Table 3.

Total content of 20 ginsenosides (mg / g) Primary Secondary Average Stdev. MeJA 6.250 6.294 6.272 0.031 MeJA2 6.495 6.369 6.432 0.090 Control group 2.268 2.080 2.174 0.133

As shown in FIG. 4, the ginsenoside content of wild ginseng stem cells was increased by> 286% as compared to the control group.

3-3. Ginsenoside analysis

The ginsenosides of callus were extracted as follows. First, weigh accurately 50 mg of Callus, place in 15 mL tube, add 5 mL of 70% methanol, and mix by shaking at 2000 rpm for 1 minute using multi-vortexer. Then, ultrasonic extraction was carried out in a water bath at 40 ° C for 1 hour, and the extract was transferred to a 2 ml tube and centrifuged at 13200 rpm for 5 minutes. Add 50 uL of clean supernatant and add 20 μL of internal standard (Digoxin 1 μg / mL, ChemFaces, China) and 100 μL of 50% methanol, mix with a simple tablet mixer, and take 5 μL of UPLC / Q-Orbitrap Fisher Scientific Inc., USA) and quantitatively analyzed by a PRM (Parallel Reaction Monitoring) method. The results are shown in FIGS. 5, 6, 4 and 5.

Percentage (%) Re 0.661 + 0.021 Rg1 9.806 + - 0.287 Rf 1.397 + 0.041 Rb1 84.162 0.022 Rc 0.222 + 0.043 Rg2 0.288 + 0.012 Rb2 0.000 占 .000 20R_Rg2 0.321 + 0.023 Rh1 0.335 0.049 20R_Rh1 0.000 占 .000 Rd 1.750 + 0.039 Gp17 0.689 ± 0.203 F2 0.059 ± 0.016 Rg3 0.087 ± 0.007 20R_Rg3 0.124 ± 0.000 Rk1 0.000 占 .000 Rg5 0.000 占 .000 CK 0.097 0.012 Rh2 0.000 占 .000 20R_Rh2 0.000 占 .000 Total 100,000

Percentage (%) Re 0.435 + 0.083 Rg1 3.897 + 0.109 Rf 0.578 + 0.013 Rb1 65.661 + 0.766 Rc 3.143 + - 0.057 Rg2 0.154 + 0.012 Rb2 2.224 0.048 20R_Rg2 0.162 0.005 Rh1 0.071 ± 0.007 20R_Rh1 0.000 占 .000 Rd 14.726 + 0.361 Gp17 6.347 + 0.264 F2 2.199 + 0.040 Rg3 0.242 ± 0.002 20R_Rg3 0.059 + 0.015 Rk1 0.000 占 .000 Rg5 0.000 占 .000 CK 0.050 0.006 Rh2 0.052 + 0.012 20R_Rh2 0.000 占 .000 Total 100,000

As can be seen in Figures 5, 6, 4 and 5, ginsenoside diol Rb1 accounted for 84.2% of total ginsenoside content and 65.7% of cells harvested after treatment with methyl jasmonate elicitor Thus, it was confirmed that the biosynthesis of ginsenoside downstream stream was remarkably progressed.

% Rg1 Rb1 Rg3 Sum MeJA2 3.9 65.7 0.2 69.8 Control group 9.8 84.2 0.1 94.1

As shown in FIG. 7 and Table 6, Rg1, Rb1, and Rg3, which are known as functional ingredients of red ginseng, were confirmed to be 94.1% in the untreated group and 69.8% in the treated group.

% Rd Gp17 F2 Sum MeJA2 14.7 6.3 2.2 23.3 Control group 1.8 0.7 0.1 2.5

As shown in FIG. 8 and Table 7, the culture extract of wild ginseng stem cells was composed of Rd, F2, and Gp17. In the untreated group, the 2.5% composition showed 23.3% in the treated group and the significantly increased biosynthesis pattern Lt; / RTI >

Rd content (ug / g) Primary Secondary Average Stdev. MeJA 850.002 869.614 859.808 13.868 MeJA2 939.914 954.105 947.010 10.034 Control group 40.315 35.831 38.073 3.171 F2 content (ug / g) Primary Secondary Average Stdev. MeJA 61.418 67.555 64.487 4.339 MeJA2 141.032 141.841 141.437 0.572 Control group 1.089 1.463 1.276 0.264 Gp17 content (ug / g) Primary Secondary Average Stdev. MeJA 207.175 205.757 206.466 1.003 MeJA2 400.112 416.123 408.117 11.321 Control group 12.369 17.322 14.845 3.502

As shown in FIG. 9 and Table 8, the content composition of the ginseng stem cell culture extract was in the order of Rd> Gp17> F2, and the sum of three molecules in MeJA2 cells was about 1.5 mg / g dry and the Rd was 0.95 mg / Of the total molecule.

Conc. (ug / ml) Rd F2 Gp17 Sum MeJA2 947.01 141.437 408.117 1496.564 Control group 38.073 1.276 14.845 54.194 % Rd F2 Gp17 Total MeJA2 24.9 110.8 27.5 27.6 Control group 1.0 1.0 1.0 1.0

10, 11 and Table 9, in the MeJA2 cells in which ginsenoside metabolism was promoted, the ginsenoside complex component RFG17 was 24.9 times (Rd), 110.8 times (F2), 27.5 times (Gp17), indicating a marked increase in F2.

% Rd Fe Gp17 MeJA2 63.3 9.5 27.3 Control group 70.3 2.4 27.4

Conc. (ug / ml) Rd F2 Gp17 Sum MeJA2 947.01 141.437 408.117 1496.564 Control group 38.073 1.276 14.845 54.194

12, 13, Table 10 and Table 11, the compositional ratios of the mass-amplified MeJA2 ginsenoside RFG17 were 63.3% (Rd), 9.5% (F2) and 27.3% (Gp17) (2.4% -> 9.5%) of F2 in the Rd and Gp17 contents was almost constant.

Ratio Rd F2 Gp17 MeJA2 6.7 1.0 2.9 Control group 29.8 1.0 11.6

As can be seen from Figs. 14, 15 and 12, the quantitative composition ratio of the ginseng stem cell culture extract was 7: 3: 1 based on F2, and thus it was confirmed that the extract was significantly enhanced as compared with the control group 30: 1: 12 there was.

Example 4. Evaluation of antioxidative, whitening and wrinkle efficacy

4-1. Antioxidant activity of wild ginseng callus (DPPH)

To each well of the 96-well plate was added 200 ul of a 0.2 mM DPPH solution. 100 μl of sample was added to each well. Vitamin C was used at concentrations of 125, 62.5, 31.25, 15.63, 7.81, 3.91, and 1.95 with the highest concentration of 125 ug / ml. For the control (control), 100 μl of distilled water was added instead of the sample. After reacting at room temperature for 10 minutes, the absorbance was measured at a wavelength of 525 nm, and the results are shown in FIG. 16 and Table 13.

Samples 50 μg / ml
Vit.C Control group Meja Meja2 Inhibition% 94.8% 84.9% 74.4% 68.3%

As can be seen from FIG. 16 and Table 13, DPPH radical scavenger activity test confirming the presence of active oxygen inhibition showed that the antioxidant activity was in the untreated group and treated group of the cultured ginseng stem cell culture extract. Antioxidants can be useful as skin whitening and wrinkle reducing raw materials.

4-2. Antioxidant activity of wild ginseng callus (SOD-like activity)

100 ul of 0.12 M Tris-HCl buffer (pH 8.0) was added to each well. 10 ul of Elastase (2.5 U / ml) was added. 20 μl of the sample was added to each well and allowed to react at 25 ° C for 10 minutes. 100 μl of 1.0 mM N-succinyl- (Ala) 3-p-nitroanilide was added to each well, followed by reaction at 25 ° C for 10 minutes. The absorbance was measured at 410 nm, and the results are shown in Fig. 17 and Table 14.

Samples 300 μg / ml
Vit.C Control group Meja Meja2 Inhibition% 50.8% 65.6% 87.8% 87.8%

As can be seen from FIG. 17 and Table 14, the SOD-like activity test confirming the presence of active oxygen inhibition showed that the antioxidant activity was in the untreated group and the treated group of the culture extract of wild ginseng stem cells. Antioxidants can be useful as skin whitening and wrinkle reducing raw materials.

4-3. Wild ginseng callus whitening effect (tyrosine naase enzyme inhibition, L-tyrosine)

Add 100 μl of mushroom tyrosinase (420 U / ml) to each well. Arbutin or 100 μl of the sample was added to each well and reacted at 37 for 10 minutes. 100 μl of L-Tyrosine solution was added to each well and reacted at 37 for 10 minutes. Absorbance values were measured at 475 nm, and the results are shown in Fig. 18 and Table 15. Fig.

Samples 600 [mu] g / ml Arbutin Control group Meja Meja2 Inhibition% 54.40% 27.6% 33.8% 32.1%

As shown in Fig. 18 and Table 15, the experiment for confirming the inhibition of tyrosinase activity, which affects melanin production, was performed. In the whitening experiment using tyrosine as substrate, the results of inhibiting tyrosinase activity in non - treated group and treated group of wild ginseng stem cell culture extract were confirmed. It can be used effectively as a whitening raw material.

3-4 Wild ginseng callus whitening effect (tyrosine naase enzyme inhibition, L-DOPA)

30 μl of mushroom tyrosinase (333 U / ml) and 10 μl of sample were added to each well, followed by addition of 60 μl of 0.1 M potassium phosphate buffer (pH 6.8). 100 μl of a 12 mM L-DOPA solution was added to each well, and the absorbance was immediately measured at 492 nm. The results are shown in FIG. 19 and Table 16.

Samples 30ug / ml Kojic acid Control group Meja Meja2 Inhibition% 51.7% 32.0% 34.2% 49.9%

As shown in Fig. 19 and Table 16, experiments for confirming inhibition of tyrosinease activity, which affect melanin production, were performed. In the whitening experiment using Eldopa as a substrate, the inhibition of tyrosinase activity was observed in the non - treated group and the treated group of the cultured ginseng stem cell culture extract. It can be used effectively as a whitening raw material.

3-5. Effect of waxy callus improvement (inhibition of elastin degrading enzyme)

100 ul of 0.12 M Tris-HCl buffer, pH 8.0 was added to each well. 10 ul of the wells elastase (2.5 U / ml) was added. 20ul of the sample was added to each well and reacted at 25 DEG C for 10 minutes. 100 μl of 1.0 mM N-succinyl- (Ala) 3-p-nitroanilide was added to each well, reacted at 25 ° C for 10 minutes, and the absorbance at 410 nm was measured. The results are shown in FIGS. 20 and 17 Respectively.

Samples 10 [mu] g / ml
(N- (Methoxysuccinyl) -Ala-Ala-Pro-Val-CMK) Control group Meja Meja2 Inhibition% 54.8% 9.6% 17.2% 15.2%

As shown in FIG. 20 and Table 17, experiments for confirming the inhibition of elastase activity, which affects wrinkle formation, were conducted. In the inhibition of wrinkle formation, the inhibition of elastin degrading enzyme activity was confirmed in the untreated group and the treated group of the cultured ginseng stem cell culture extract. Can be usefully used as wrinkle improving raw material

3-6. Effect of improving the wrinkle of ginseng callus (hyaluronic acid degradation enzyme inhibition)

The enzyme dissolved in 0.1 M acetate buffer (pH 3.5) and 6 ul of HAase (10 mg / ml) were added to each well of a 96-well plate. The mixture was mixed with 6 μl of the sample and reacted in a 37 incubator for 20 minutes. To activate the HAase, 6 μl of 12.5 mM CaCl 2 was added to the mixed solution, and the mixture was incubated in a 37 ° C incubator for 20 minutes. After the reaction, 24 μl of the substrate solution hyaluronate (3 mg / ml) dissolved in 0.1 M acetic acid buffer solution (pH 3.5) was added to the reaction solution, which was reacted for 40 minutes in a 37 incubator. Add 6 μL of 0.4 N NaOH and 6 μL of 0.4 M potassium tetraborate to the reaction mixture and react at 100 ° C for 3 minutes. After completely cooling at room temperature, centrifuge at 1500 rpm for 2 minutes. After adding 180 μl of DMAB solution, the absorbance at 540 nm was measured using an ELISA reader. The results are shown in FIG. 21 and Table 18.

Samples 1000 μg / ml
Tannic acid Control group Meja Meja2 Inhibition% 78.30% 0.0% 44.3% 40.9%

As shown in FIG. 21 and Table 18, experiments for confirming the inhibition of hyaluronic acid degrading enzyme activity, which affects wrinkle formation, were conducted. The inhibition of hyaluronic acid degrading enzyme activity was not observed in the untreated group of wild ginseng stem cell culture extract, but it was confirmed that the treatment group inhibited hyaluronic acid degrading enzyme activity. It can be usefully used as a wrinkle improving raw material.

Claims (15)

Obtaining a wild ginseng callus in which a Korean wild ginseng slice is tissue-cultured in an MS (Murashige and Skoog) medium supplemented with sucrose, 2,4-dichlorophenoxy acetic acid and gelite;
Culturing suspension cells in which MS ginseng callus is treated with methyl jasmonate or methyl jasmoic acid in an MS medium supplemented with sucrose and 2,4-dichlorophenoxyacetic acid to culture floating cells; And
Removing the culture medium, mixing distilled water, and freeze-drying using a freeze dryer;
Which comprises culturing the ginseng stem cell culture extract. The method according to claim 1, wherein the step of obtaining the ginseng callus is tissue culture in an MS (Murashige and Skoog) medium supplemented with 3% sucrose, 1 mg / L 2,4-dichlorophenoxyacetic acid and 2.4 g / L gelite A method for producing an extract of cultured human ginseng stem cell. The method according to claim 1, wherein the floating cell culture is performed with a bioreactor or a Rotary Cell Culture System (RCCS). The method according to claim 1, wherein said methyl jasmonate or methyl jasmonate is treated with 200 uM. The method according to claim 1, wherein the floating cell culturing step is performed for 10 to 30 days. The method according to claim 1, wherein the ginsenoside cell culture extract has a ginsenoside concentration of 6 mg / g or more by LC / Q-Orbitrap assay. [Claim 3] The method according to claim 1, wherein the total concentration of saponin in the culture extract of wild ginseng stem cells is 1% to 3% by a sulfuric acid-vanillin chromogenic assay using an absorbance meter. [Claim 2] The method according to claim 1, wherein the ginseng stem cell culture extract has a biosynthesis weight ratio of Rg1, Rb1, and Rg3 of 60 to 80% of total ginsenoside weight. [Claim 3] The method according to claim 1, wherein the ginseng stem cell culture extract has a total ginsenoside content of 5.0 to 8.0 mg / g. Ginseng stem cell culture extract having a total saponin content of 20.5 mg / g or more and a total ginsenoside content of 6.0 mg / g or more. [Claim 11] The cultured ginseng stem cell culture extract according to claim 10, wherein the content of Rd, F2 and Gp17 contained in the cultured ginseng stem cell culture is 10-30% of the total ginsenoside content. [Claim 11] The cultured ginseng stem cell culture extract according to claim 10, wherein the weight ratio of Rd: Gp17: F2 in the cultured ginseng stem cell culture is 7: 3: 1. A cosmetic composition for improving skin condition comprising an extract of wild ginseng stem cell culture. The cosmetic composition according to claim 13, wherein the skin condition is improved wrinkles, skin elasticity, skin aging, skin moisturizing, acne improvement or whitening. A pharmaceutical composition for prevention of skin aging comprising an extract of cultured ginseng stem cell.

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