KR101593905B1 - Method for promoting secondary metabolites of wild ginseng cell - Google Patents

Abstract

The present invention relates to a method for promoting the production of secondary metabolites of wild ginseng cells. In one embodiment, the method for promoting the production of secondary metabolites of wild ginseng cells comprises the steps of inducing embryogenic callus from wild ginseng cotyledons; Culturing the embryogenic callus to induce somatic embryo cells; And culturing the somatic cell-culturing cells at a pH of 4.5 to 6.0 and a temperature of 10 to 25 ° C .; and culturing the somatic cells by ultrasonic stimulation and light stimulation.

Description

METHOD FOR PROMOTING SECONDARY METABOLITES OF WILD GINSENG CELL [0002]

The present invention relates to a method for promoting the production of secondary metabolites of wild ginseng cells. More specifically, the present invention relates to a method for stimulating and culturing somatic embryo-derived cells of wild ginseng without using chemical substances, thereby preventing the accumulation of chemical substances in the cells and improving the content of active ingredients of wild ginseng cells And a method of promoting generation.

The wild ginseng ( Panax ginseng CA Meyer or Panax schinseng NEES) is a plant that is native to the Far East region of Asia. It is located in Korea (33.7 ° ~ 43.1 °) excluding Jeju Island, (43 ° ~ 47 °) in the Manchurian region (40 ° ~ 48 °) in Russia, and in the coastal region of Russia (40 ° ~ 48 °).

The wild ginseng has been used medicinally from ancient times and has been recorded as a medicinal material in many treatments including Dongbokgwon, mushrooms, Maternal Maternity, Divine Materia Medica, Meanwhile, recent studies have shown anticancer effects, blood glucose lowering, and prevention of obesity. Studies on toxins, safety tests and in vitro tests related to herbal medicine have been reported as research using goat ginseng.

The wild ginseng has a high selectivity for the cultivation area such as dormancy or growth rate is very slow in places where the natural environment such as climatic soil is not suitable, so it is difficult to grow and the wild ginseng is already depleted due to beeswax. It is known that the breeding power is very weak because it takes longer than a year.

Techniques for mass production of adventitious roots using plant tissue culture technology have been actively researched for these rare and high added value ginsengs, and efforts have been made to mass produce them in a short period of time and use them in various foods and raw materials. However, in the conventional methods using plant tissue culture techniques, callus, which is undifferentiated cell, is cultured in a biological incubator, or adventitious roots are induced to use wild ginseng adventitious roots or extracts thereof. Especially, There is little research to induce promotion.

It has been known that when callus is formed from somatic cells of wild ginseng and differentiated into adventitious roots, plant growth regulators accumulate in the adventitious roots, making it difficult to grow uniformly in the latter stages of culture.

Secondary metabolites, on the other hand, do not have the primary and direct physiological functions necessary for survival, such as growth and development of plants, and are additional natural products that are limited to specific plants. These secondary metabolites are distinguished from primary metabolites such as sugar phosphates, amino acids, amides, nucleotides, nucleic acids, and organic acids that are essential for plant life. Animal is motile and can escape from external threats, while plants without motility can protect themselves from pests and pathogens by creating toxic substances through self-defense measures, and the contention of nutrients among plants or the breeding of species To produce secondary metabolites by means of attracting insects and the like. Since these secondary metabolites act mostly as physiologically active substances, interest is growing. (Reported Plant Tissue Culture, Experiment, Fracture)

On the other hand, ginsenoside refers to saponin which is a secondary metabolite of wild ginseng. Saponin is derived from Greek word meaning soap, and is named as a soap-like fine bubble in aqueous solution. It is a kind of compound called chemically glycoside, in which at least one sugar molecule is attached to the non-sugar moiety of the pin. Recently, anticancer, antioxidant, and cholesterol lowering effects have been discovered, and they have been attracting attention as physiologically active substances and have been used as a cardiotonic or diuretic in oriental medicine. Especially, saponin plays a main pharmacological role among various active ingredients of wild ginseng. Saponin of this wild ginseng has a unique chemical structure different from that of saponin found in other plants. Its pharmacological effect is also unique and is called ginsenoside. In the case of ginseng, ginseng A much greater amount of ginsenoside is included.

These ginsenosides are named ginsenoside-Rx, where "R" stands for Radix or Root Root, and X for Sample spot on TLC (Thin Layer Chromatography) A, b, c, d, e, f, g, and h are ordered from bottom to top according to the numerical order (Shibata et al., 1966). Ginsenoside is a triterpenoid saponin, which is classified into dammarane saponin and oleanane saponin. The saponin of the fumarion system is composed of protopanaxadiol (PPD) saponin and protopanaxatriol, depending on the number of hydroxyl groups (-OH) attached to the aglycone having tetrahedral fumarate skeleton. PPT) saponins (Matsuura et al., 1984; De Smet, 2004).

The PPD-based saponin has two hydroxyl groups (positions C3 and C20), and the PPT family has three hydroxyl groups (positions C3, C6, and C20). These saponins have an ester bond in the hydroxyl group with saccharides such as glucose, rham, xyl, arabinose and the like.

The PPD type ginsenosides are ginsenosides-Ra1, -Ra2, -Ra3, -Rb1, -Rb2, -Rb3, -Rc, -Rd, -Rs1, -Rs2, -Rs3, -Rg3 and Rh2, The PPT family ginsenoside is ginsenoside-Re, -Rf, -Rg1, -Rg2, -Rh1, -Rh4, and the like.

In addition, the wild ginseng contains phenolic compounds and flavonoids as secondary metabolites. The phenolic compounds exhibit antioxidant activity, exhibit anticholesterol, suicide action, anticancer and sulfation (Du val and Shetty, 2001) The flavonoids are classified into flavonols, flavones, catechins and iso-flavones depending on the chemical structure and are known to have an effect such as suppression of lipid peroxidation (Middleon and Kandaswami, 1994).

On the other hand, it has been known that when various stresses (or stimuli) are applied to cultivate wild ginseng cells that are not induced into adventitious roots, the generation of corresponding secondary metabolites increases, and as a elicitor, Or a method of promoting the production of secondary metabolites by treating a precursor of a secondary metabolite to a medium.

Korean Patent Laid-Open Publication No. 2005-0102571 discloses a method of inducing callus by inducing wild ginseng to a solid medium for callus induction under dark conditions; Culturing the callus in a liquid culture medium for callus suspension culture and shaking until the total amount of the liquid medium is 25%, and culturing the callus in the form of a single cell; The elicitor and saponin such as jasmonic acid, yeast extract, chitosan, ferulic acid and caffeic acid are added to the culture medium of the electrocultured callus. Adding a precursor; And shaking culturing the callus until all of the sugar in the medium to which the precursor of the electric cleanser and the saponin is added is consumed to obtain wild ginseng cells.

However, when the artificial chemical is used as the illiterator or when the precursor is applied, the production of the secondary metabolites of the wild ginseng cells can be promoted, but since the artificial chemical is absorbed and accumulated in the cells, There is still a problem that it is not certain that the efficacy is due to secondary metabolites of wild ginseng cells.

It is an object of the present invention to provide a method for promoting the production of secondary metabolites of wild ginseng cells which do not accumulate plant growth hormone and chemical substances in cells, are cytotoxic, and harmless to humans.

Another object of the present invention is to provide a method for promoting the production of secondary metabolites of wild ginseng cells, which is excellent in the treatment, improvement, prevention and alleviation effect of inflammation, antioxidative effect, whitening and skin tone improvement effect.

It is still another object of the present invention to provide wild ginseng cells prepared by the method for promoting the production of secondary metabolites of wild ginseng cells.

It is still another object of the present invention to provide a cosmetic composition comprising the ginseng cell or an extract thereof.

One aspect of the present invention relates to a method for promoting the production of secondary metabolites of wild ginseng cells. In one embodiment, the method for promoting the production of secondary metabolites of wild ginseng cells comprises the steps of inducing embryogenic callus from wild ginseng cotyledons; Culturing the embryogenic callus to induce somatic embryo cells; And culturing the somatic cell-culturing cells at a pH of 4.5 to 6.0 and a temperature of 10 to 25 ° C .; and culturing the somatic cells by ultrasonic stimulation and light stimulation.

In one embodiment, the ultrasound stimulation may be performed by treating the somatic cells with a frequency of 20 to 50 kHz, and irradiating the somatic cells with light with a wavelength of 380 to 430 nm.

In one embodiment, the average content of the phenolic compounds contained in the wild ginseng cells is 0.115-0.219 mg / g, the average content of flavonoids is 0.18-0.28 mg / g, and the average content of ginsenosides is 5.85 To 8.65 mg / g.

The method of promoting the production of secondary metabolites of wild ginseng cells of the present invention uses a somatic embryo not differentiated into an organ such as a stem or a root to prevent accumulation of a plant growth hormone in a cell during culturing and to use a chemical as an illiterer And the cosmetic composition comprising the ginseng cell or the extract thereof produced by the above production method is effective for the treatment, improvement, prevention and alleviation of inflammation , Antioxidant effect, whitening effect and skin tone improvement effect.

1 is a photograph of HPLC analysis of wild ginseng cells prepared according to one embodiment of the present invention.

In the following description of the present invention, a detailed description of known functions and configurations incorporated herein will be omitted when it may make the subject matter of the present invention rather unclear.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed.

One aspect of the present invention relates to a method for promoting the production of secondary metabolites of wild ginseng cells. In one embodiment, the method of promoting the production of secondary metabolites of wild ginseng cells comprises the steps of: (a) inducing the cotyledon of the wild ginseng; (b) inducing embryogenic calli; (c) inducing somatic cell embryonic cells; And (d) a somatic cell culture cell culture step.

More specifically, the method for promoting the production of secondary metabolites of wild ginseng cells comprises the steps of inducing embryogenic callus from cotyledons of wild ginseng; Culturing the embryogenic callus to induce somatic embryo cells; And culturing the somatic embryo-cultured cells, wherein the somatic embryo-derived cells are stimulated using ultrasound and optical stimulation during the culturing.

Hereinafter, the method for promoting the production of secondary metabolites of wild ginseng cells of the present invention will be described in detail.

(a) induction of cotyledon of wild ginseng

This step induces immature cotyledons through germination of aseptic germination of wild ginseng seeds. In one embodiment, the dormancy of the ginseng seeds can be broken, the ginseng seeds can be disinfected, and then the immature cotyledons can be induced through sterile germination.

The seeds of the wild ginseng were treated with a low temperature layer treatment for 5 to 7 months to break the dormancy. Then, the seeds of the ginseng seeds were removed and sterilized in 70% ethanol for 30 seconds to 1 minute, and 1% sodium hypochlorite sodium hypochloride solution for 20 to 40 minutes and sterilized with distilled water three to four times.

The low-temperature layered treatment may be carried out by a conventional method. In the specific example, when the above-mentioned ginseng seeds are kept at a humidity within the range of not drying, they are mixed with the sand for 2 to 4 months to store the ginseng seeds, and the ginseng seeds after the openings are subjected to low temperature treatment for 2 to 4 months (Around 10 ° C), so that dormancy can be broken.

The sterilized ginseng seeds may be seeded in sterilized glass dishes on a sterilized plate and cultured aseptically at 22 ° C to 38 ° C to induce immature cotyledons.

The medium may be a conventional one, for example, a 1/2 MS (Murashige & Skoog) medium may be used, but is not limited thereto.

(b) induction of embryogenic callus step

This step induces embryogenic callus from the cotyledons of the derived wild ginseng. In the present invention, premature cotyledons immediately before germination can be used for callus induction.

In the present invention, the embryogenic callus induction medium may be any conventional one. For example, B5 (Gamborg's B5), Durzan, Murashige & Skoog, SH, Schenk & Hildebrandt, White, DKW, Gresshoff & Doy, &Amp; Lepiovre) or WPM (woody plant medium) medium.

Plant growth hormones such as 2,4-D (2,4-dichlorophenoxyacetic acid) contained in the WPM and the like are used for culturing somatic cell cultured cells described later under temperature and pH culture conditions of the present invention , And it is hardly contained in wild ginseng cells produced by the production method of the present invention.

In an embodiment, the immature cotyledons of the wild ginseng are cultured in WPM solid containing 0.25% gelrite, 3% sucrose and 0.1-5 ppm, preferably 1 ppm to 1.5 ppm, of 2,4-D The callus can be induced by dorsal contact with the medium. In this case, the embryogenic callus of the wild ginseng can be induced by controlling the photoperiod of the wild ginseng at 24 ° C to 28 ° C for 20 hours with light of 20-30 μmol · photons / m ² / s for 16 hours,

Embryogenic callus may be induced in subculture 2-3 or 4 weeks apart under the same culture conditions.

(c) somatic embryo cell induction step

The step is to induce somatic embryogenic cells by culturing the embryogenic callus. The embryogenic callus may be subcultured four or more times at intervals of two weeks to proliferate the cells. After 8 weeks, somatic embryonic cells may be induced by subculturing at intervals of four weeks.

(d) somatic cell culturing step

This step is a step of culturing somatic cell-derived cells. In one embodiment, the somatic embryo-cultured cells can be cultured at pH 4.5 to 6.0 and 10 to 25 ° C for 2 to 6 weeks. Preferably at a pH of 5.0 to 5.5 and at a temperature of 10 to 20 DEG C for 2 to 6 weeks. When cultured under the above-mentioned pH and temperature conditions, the amount of secondary metabolites of the wild ginseng cells of the present invention is promoted and the somatic embryo cells are stimulated to undergo physiological stress without being killed in the pH or temperature range. To increase the total amount of synthesis of secondary metabolites. When cultured under the above-mentioned pH and temperature range, the ginseng cells may not grow or die.

In another embodiment, somatic embryogenic cells are cultured at pH 4.5 to 7.0 and 20 to 30 ° C for 2 to 3 weeks and then cultured at pH 4.5 to 6.0 and 10 to 25 ° C for 2 to 6 weeks have. Under these conditions, wild ginseng cells can be easily grown and the amount of secondary metabolites produced can be promoted.

In the present invention, a medium for culturing somatic cell-seeding cells can be used. For example, B5 (Gamborg's B5), Durzan, Murashige & Skoog, SH, Schenk & Hildebrandt, White, DKW, Gresshoff & Doy, &Amp; Lepiovre) or WPM (woody plant medium) medium.

In addition, somatic embryo-cultured cells can be stimulated by using ultrasonic stimulation and light stimulation during the culturing. The amount of the secondary metabolites of the wild ginseng cells of the present invention can be further promoted during the stimulation using the ultrasonic stimulation and the light stimulation.

In one embodiment, the ultrasound stimulation is performed by treating the somatic embryo-coated cells at a frequency of 20 to 50 kHz for 20 to 60 minutes, and the photostimulation is performed by irradiating the somatic cells with a wavelength of 380 to 430 nm for 10 to 120 minutes You can investigate.

In a specific example, ultrasonic stimulation and light stimulation under the above conditions can be stimulated once or twice a day during the incubation period of the somatic embryogenic cells. The amount of the secondary metabolites of the wild ginseng cells of the present invention can be further promoted upon stimulation under the above conditions.

In the present invention, ultrasound stimulation and light stimulation may be performed simultaneously or alternately when culturing the somatic cells.

As described above, the plant growth hormone such as 2,4-D contained in the medium such as WPM is degrade by the light stimulation and the ultrasonic stimulation under the temperature and pH culture conditions, There is an advantage that it is hardly contained in wild ginseng cells produced by the method.

As described above, when the method for promoting the production of secondary metabolites of wild ginseng cells is applied, it is possible to provide an environment for promoting the production of secondary metabolites of wild ginseng cells without using chemicals.

Another aspect of the present invention relates to wild ginseng cells prepared according to the method for promoting the production of secondary metabolites of wild ginseng cells. In one embodiment, the ginseng cell may comprise a phenolic compound, a flavonoid, and a ginsenoside.

The phenolic compound exhibits an antioxidant activity effect, and can exhibit whitening, anti-inflammation and antioxidant activity.

The flavonoids may exhibit whitening, anti-inflammatory and antioxidant activities. The flavonoids may include flavonols, flavones, catechins and iso-flavones, and the like.

The ginsenosides can enhance the anti-inflammatory, whitening and antioxidative effects of the present invention. The gensenoside of the present invention may be a ginsenoside-Rb1, -Rb2, -Rb3, -Rc, -Re, -Rf, -Rg1, -Rg2, -Rg3, -Rh2, -F1, -F2, PPD, -PPT, and -Compound-K, and the like

In one embodiment, the average content of the phenolic compounds contained in the wild ginseng cells is 0.115 to 0.219 mg / g (or 0.115 to 0.219 mg / ml) and the average content of flavonoids is 0.18 to 0.28 mg / g (or 0.18 to 0.28 mg / ml), and the average content of ginsenosides may be 5.85 to 8.65 mg / g (or 5.85 to 8.65 mg / ml). Within the above range, the anti-inflammatory, whitening and antioxidative effects of the present invention can be excellent.

Another aspect of the present invention relates to a cosmetic composition comprising the extract of the wild ginseng cell. In the present invention, the wild ginseng cell extract may be obtained by extracting the wild ginseng cells with an organic solvent.

The organic solvent extraction may be carried out from a mixture comprising 100 parts by weight of the ginseng cells and 100-2000 parts by weight of the organic solvent. Under these conditions, the oil component contained in the wild ginseng cells can be easily extracted without being damaged.

Examples of organic solvents that can be used in the organic solvent extraction include acetone, ethyl acetate, diethyl ether, glycerin, ethylene glycol, propylene glycol, butylene glycol, benzene, chloroform, hexane and alcohols having 1 to 4 carbon atoms . These may be used alone or in combination of two or more. When the organic solvent as described above is used, the oil component contained in the wild ginseng cells can be easily extracted without being damaged.

As the alcohol, for example, intermediate divalent alcohols such as lower monohydric alcohols such as methanol, ethanol and butanol, lower trihydric alcohols such as propylene glycol, 1,3-butylene glycol and glycerin, and the like can be used. These may be used alone or in combination of two or more.

In one embodiment, the organic solvent may be a mixture of ethylene glycol, ethanol, and ethyl acetate in a volume ratio of 1: 0.1-5: 0.1-5. Using the organic solvent of the mixing ratio, the useful ingredient contained in the wild ginseng cells is extracted and fractionated, and then concentrated under reduced pressure or dried to obtain a ginseng cell extract. As described above, useful components of the wild ginseng cell can be effectively extracted during the extraction.

In one embodiment, an extractor with a cooling condenser may be used to prevent organic solvents from evaporating during organic solvent extraction. In another specific example, the active ingredient may be extracted by immersion at 5 to 30 ° C for 1 to 7 days, followed by concentration with a reduced pressure concentrator.

In the present invention, the ginseng cell extract may be contained in an amount of 0.01 to 50% by weight based on the total weight of the cosmetic composition. Preferably 0.1 to 30% by weight. More preferably 1 to 10% by weight. In the above-mentioned range, it is possible to have an excellent effect of treating, improving, preventing and alleviating inflammation, antioxidative effect, whitening and improving skin tone without skin irritation and cytotoxicity.

In addition, the cosmetic composition may further comprise a base component which can be compounded in a conventional cosmetic composition. The specific examples may further include a solubilizer, a surfactant, a moisturizer, an agitator, a pH adjuster, a preservative, an antioxidant, a sequestering agent, a bactericide, an anti-inflammatory agent, an antimicrobial agent, a solubilizing agent or a flavoring agent, .

Examples of the surfactant that can be included in the composition include anionic surfactants such as ammonium laurylsulfosuccinate and ammonium lauryl sulfate and nonionic surfactants such as polyoxyethylene alkyl ether and polyoxyethylene fatty acid ester . Also, cationic surfactants such as tertiary aliphatic amine salts, alkyltrimethylammonium chloride and the like and amphoteric surfactants such as betaine, amide betaine and the like can be used.

The moisturizing agent may be sodium hyaluronate, glycerin, propylene glycol, 1,3-butylene glycol, dipropylene glycol, sorbitol, etc. The thickening agent may be xanthan gum, methyl cellulose, hydroxyethyl cellulose, carrageenan, carboxy Methylcellulose, hydroxymethylcellulose, and other water-soluble polymeric compounds. The pH adjuster may specifically include citric acid, sodium hydroxide, triethanolamine, sodium citrate, phosphoric acid, sodium phosphate, lactic acid, and the like.

The preservative may be benzoic acid, paraoxybenzoic acid ester, a mixture of methylchloroisothiazolinone, phenoxyethanol, DMDM, and the like. Specific examples of the antioxidant include dibutylhydroxytoluene and ascorbic acid .

The metal ion sequestering agent may be disodium ethylenediaminetetraacetate, tetranodium ethylenediaminetetraacetate, and the like. Specifically, the disinfectant may be chlorhexidine gluconate, quaternary ammonium salt, pyrrothonol amine, zinc pyrithione suspension, urethropropylbutyl Carbamate, salicylic acid, and the like.

Examples of the anti-inflammatory component include monoammonium glycyrrhizinate, dipotassium glycyrrhizinate, allantoin, and mixtures thereof. The antibacterial (antimicrobial) component specifically includes phenoxyethanol, chlorhexidine, chlorhexidine gluconate, benzoic acid and its salts, benzyl Alcohols, salicylic acid, trichlorocarbane, zinc pyrithione suspensions, and mixtures thereof.

The solubilizing agent may specifically be a phage 60 -hydrogenated castor oil or the like, and the above-mentioned flavoring agent may be any conventional agents used in the cosmetic composition field of the present invention.

In addition, the cosmetic composition of the present invention may be prepared in any form conventionally produced in the art, and examples thereof include a skin, a lotion, a gel, a cream, a massage cream, an essence, a foundation, a powder, a mascara, Eye shadow, lipstick, pack, soap, foam cleansing, cleansing cream, sea lotion, body cleanser, hair cosmetic composition.

Hereinafter, the configuration and operation of the present invention will be described in more detail with reference to preferred embodiments of the present invention. However, the following examples are provided to aid understanding of the present invention, and the scope of the present invention is not limited to the following examples. The contents not described here are sufficiently technically inferior to those skilled in the art, and a description thereof will be omitted.

Example 1: Production of wild ginseng cells

Seed disinfection of wild ginseng and in-flight germination induction

Methods for seed sterilization and in vitro germination of wild ginseng are as follows. Germination of wild ginseng seeds is slowed down by low temperature layer treatment for about 6 months. After removing the seeds of seeds that had been dormant, the seeds were sterilized in 70% ethanol for 30 seconds to 1 minute, sterilized in 1% sodium hypochlorite solution for 20 minutes or more, and rinsed 3 times to 4 times with sterilized distilled water. The surface-sterilized seeds were divided into 1 / 2MS medium supplemented with 2% sucrose and 0.25% gelrite, and then cultured in the dark. The seeds immediately before germination were used for callus induction.

Inducing embryogenic callus and somatic embryo cell

The obtained wild ginseng cotyledon was cultured in a WPM solid medium containing 0.25% gelite, 3% sucrose and 2,4-D at 1 ppm to 1.5 ppm to induce callus. The incubator for callus induction was adjusted to 24 ° C and the photoperiod was regulated by light treatment for 16 hours and dark treatment for 8 hours under illumination of 24 μmol · photons / m ² / s. The cotyledon of the wild ginseng is in contact with the medium to induce callus. The ginseng callus is subcultured four times or more at intervals of 2 weeks, and cells are proliferated. After 8 weeks, somatic embryonic cells are induced by subculturing at intervals of 4 weeks.

Somatic cell cultured cell culture

The method of isolating the cultured cells from each other by liquid suspension culture of the derived somatic embryo-derived cells of the wild ginseng is as follows. 200 ml of WPM liquid medium containing 3% sucrose without gel light and plant growth regulator was prepared in a disrupted 500 ml glass Erlenmeyer flask. At this time, the pH of the WPM liquid medium was 5.7.

5 g of somatic cell body weight of wild ginseng was inoculated on the WPM liquid medium, sealed with disinfected aluminum foil, and stirred at 120 rpm in a stirrer. The incubation temperature for the liquid suspension culture was adjusted to 10 ℃ and the photoperiod was regulated by light treatment for 16 hours and dark treatment for 8 hours under illumination of 24 μmol · photons / m ² / s. After incubation for 2 weeks, And incubated for an additional 2 weeks while stimulating the somatic embryos of the wild ginseng. The stimulation was repeated for 2 weeks with ultrasonic stimulation at a frequency of 30 kHz and light stimulation at a wavelength of 400 nm for 80 minutes once a day, respectively.

Comparative Example 1

Wild ginseng cells were prepared in the same manner as in Example 1 except that ultrasonic stimulation was not applied.

Comparative Example 2

Wild ginseng cells were prepared in the same manner as in Example 1 except that no optical stimulus was applied.

Comparative Example 3

Wild ginseng cells were prepared in the same manner as in Example 1, except that somatic cell-cultured cells were cultured by adjusting the pH of the WPM liquid medium to 6.5.

Comparative Example 4

The ginsenoside cells were prepared in the same manner as in Example 1, except that the somatic embryo-cultured cells were cultured at a temperature of 28 ° C for the liquid suspension culture.

Experimental Example 1: Cell activity test

The WST assay measures the mitochondrial dehydrogenase activity in cells by measuring live cells. WST is an aqueous tetrazolium salt that reacts with living cells to produce an orange-soluble formazan. This formazan is measured to confirm the cytotoxicity of the sample.

The toxicity of macrophages (RAW 264.7) was measured in order to confirm that the water of the present invention can exhibit an immunological activity effect without irritation such as inflammation exacerbation and the like. Macrophages were counted on a 96-well plate using a hemacytometer in the same manner as 1 × 10 ⁴ cells / well. The embodiment of the present invention to a macrophage culture for 24 hours in Examples 1 to 3 and Comparative Examples 1 ~ 4 0.1 (v / v )% into each well, 37 ℃ 100㎕ in DMEM medium, at 5% CO ₂ incubator 24 Lt; / RTI > After the supernatant was removed, DMEM medium and WST solution (EZ-cytox) were mixed at a ratio of 9: 1, reacted at 37 ° C in a 5% CO ₂ incubator for 2 hours and absorbance was measured at 450 nm. Cell viability was expressed as a percentage (%) based on the absorbance of the control using a solvent such as an extract. The cell activity (%) of Example 1 and Comparative Examples 1 to 4 is shown in Table 1 below.

division Cell activity (%) Control group 100 Example 1 101.3 Comparative Example 1 101.4 Comparative Example 2 101.5 Comparative Example 3 102.2 Comparative Example 4 101.2

As shown in Table 1 and FIG. 1, in Example 1, cell activity was similar to that of the control group, and no cytotoxicity was observed.

Experimental Example 2: Antioxidant Experiment

Free radicals are active oxygen, and the oxygen that we breathe is thousands of times higher in the process of making energy and reducing it to water. It is produced by the metabolic processes of cells in plants and animals, or by stress, light stimulation, and bacterial infiltration. When excessive amount of active oxygen is present in the body, normal cells become indiscriminate attacks, diseases and aging. In addition, it acts as a pathological factor showing an immune response sensitive to external stimuli. Whether the antiinflammatory composition of the present invention exhibited antioxidant activity and had an inflammation-reducing effect was confirmed as a free radical scavenging ability of DPPH.

1 mM DPPH (1,1-diphenyl-2-picryl hydrazyl, Sigma D9132-1G, USA) generates free radicals in ethanol. 0.5 μl of each of Example 1 and Comparative Examples 1 to 4 was added to 0.25 μl of ethanol and 200 μl of 100 μM DPPH. Stirred strongly for 10 seconds, stored in a dark place for 30 minutes, and then absorbed at 517 nm. At this time, as a negative control group, the absorbance was measured using a medium in which the extract of somatic embryos of wild ginseng was not added, and the antioxidant effect was calculated by the following formula 1.

[Formula 1]

Free radical scavenging ability (%): (100- (sample treatment group / sample no treatment group)) X 100

division Free radical scavenging ability (%) Example 1 59.6 Comparative Example 1 38.9 Comparative Example 2 31.6 Comparative Example 3 39.2 Comparative Example 4 38.5

As shown in Table 2, Example 1 showed excellent antioxidative ability as compared with the control group and Comparative Examples 1 to 4.

Test Example 3. Nitric oxide (NO) production experiment

In order to measure the antiinflammatory activity of the prepared Example 1 and Comparative Examples 1 to 4, experiments were conducted to measure the concentration of nitric oxide (NO) produced by the inflammatory reaction. Stimulation of macrophages with LPS (lipopolysaccharide) activates several pathways, and NO is generated by NOS (NO synthase). The amount of NO produced was measured in the form of NO ^{2 -} present in the cell culture medium, using NO kit (Nitric oxide detection kit, iNtRON 21021, Korea).

Example 1 and Comparative Examples 1 to 4 were each dissolved in a solvent and then diluted to an appropriate concentration to prepare a stock solution. The cells were treated with macrophages (RAW 264.7 cells) with 1 μg / ml of LPS (Sigma, St. Louis, MOLOGAN, UT, USA) and cultured for 24 hours. After taking 100 μl of the culture, 50 μl of NO kit's N1 buffer was added and allowed to stand for 10 minutes. Then, 50 μl of N2 buffer was added and left for 10 minutes, and the absorbance was measured at 540 nm. NO ^{2 -} was measured using nitrite as a standard solution. In addition, when the macrophages not treated with LPS were used as a positive control group and the macrophages treated with LPS were used as a negative control, the NO ² -generating concentrations after the treatment of Example 1 and Comparative Examples 1 to 4 were compared, Respectively.

division NO production concentration (μM) Positive control group 1.0 Negative control group 15.0 Example 1 10.5 Comparative Example 1 4.5 Comparative Example 2 3.2 Comparative Example 3 3.7 Comparative Example 4 3.4

Referring to Table 3, it was found that the NO production reduction effect of Example 1 was relatively superior to that of Comparative Examples 1 to 4.

Experimental Example 4: Effect of inhibiting melanin formation

Stimulation of melanocytes with MSH activates several pathways and increases tyrosinase expression, resulting in melanin production. The resulting melanin is present in the cell culture medium and cells, and the pellet of the culture medium and the cells becomes black due to melanin.

(Melanocyte-Stimulating Hormone), which is a melanin-inducing substance, was treated in order to compare melanin formation inhibitory effects of the above-mentioned Example 1, Comparative Examples 1 to 4 and arbutin (α-arbutin, Comparative Example 5) An experiment was conducted to measure the melanin produced in one melanocyte.

B16-BL6 melanoma cells (Murine B16-F10 melanoma cells were obtained from ATCC (Manassas, Va., USA)) were cultured in a 100 mm culture dish for 24 hours and then cultured in hyclone DMEM SH30284.01 medium The extracts of somatic embryogenic cells of each wild ginseng obtained in Examples 1 to 5 were treated. MSH was used for induction of melanin. The cells were incubated with the extract for 60-72 hours, harvested by pipetting, centrifuged at 1500 rpm for 5 minutes, and the culture was dispensed into a 96-well plate at 450 nm absorbance. At this time, the culture medium in which hyclone DMEM SH30284.01 medium was used as a negative control group was used, and a culture medium in which MSH was 50nM in hyclone DMEM SH30284.01 medium was used as a positive control group.

division Melanin contents (% of control) Positive control group 100 Negative control group 220.7 Example 1 168.3 Comparative Example 1 133.5 Comparative Example 2 137.4 Comparative Example 3 130.7 Comparative Example 4 123.2 Comparative Example 5 125.4

Referring to Table 4, it can be seen that Example 1 has relatively better melanin formation inhibitory effects than Comparative Examples 1 to 4 and Arbutin (Comparative Example 5), which is known as an excellent whitening substance.

Experimental Example 5. Measurement of physiologically active substance content of wild ginseng cells

In order to examine the effect of the wild ginseng cells of the examples shown in the above Test Examples 1 to 4 on the components, the physiologically active substances of the wild ginseng cells of Example 1 and Comparative Examples 1 to 4 (total phenolic compound ) And flavonoids) were measured. At this time, as a control group, a medium in which cells were not cultured was used. Folin &Ciocalteu's Phenol Reagent was used to measure total phenolic compounds and flavonoid contents. (+) - catechin hydrate, Naringin, diethyleneglycol and Na ₂ CO ₃ were used as standards.

Test Example 5-1: Measurement of phenolic compound content

(+) - Catechin hydrate was prepared at a concentration of 0.0016-0.2 mg / ml and used as a standard reagent for the measurement of the content of total phenolic compounds in the wild ginseng cells of Example 1 and Comparative Examples 1 to 4. To each sample was added 2 % Na ₂ CO ₃ and 50% folin reagent were added at concentrations ranging from 0.0016 to 0.2 mg / ml. A standard curve was prepared by measuring at 750 nm wavelength with a UV spectrophotometer. 100 mg of lyophilized callus obtained by applying Coordinated media was taken in a 15 ml conical tube, and then 7 ml of 50% methanol was added, and the mixture was extracted with an ultrasonic extractor for 1 hour and then diluted to 10 ml with 50% methanol. The extract was filtered and used as a test solution. 100 μl of the sample solution was mixed with 2 ml of 2% Na ₂ CO _3, and 100 μl of a 50% folin reagent was added to the solution. The mixture was allowed to stand at room temperature for 30 minutes and then measured at 750 nm by UV spectrophotometer.

division Wavelength (nm) Transmittance Absorbance Concentration (mg / ml) catechin 0.002 (mg / ml) 750 88.93 0.046 0.002 catechin 0.02 (mg / ml) 750 77.36 0.111 0.023 catechin 0.2 (mg / ml) 750 23.41 0.631 0.206 Example 1 750 25.65 0.596 0.201 Comparative Example 1 750 52.53 0.125 0.109 Comparative Example 2 750 46.15 0.295 0.149 Comparative Example 3 750 37.43 0.309 0.115 Comparative Example 4 750 35.66 0.411 0.172

Referring to Table 5, Example 1, which showed excellent results in the antioxidative, anti-inflammatory and whitening effects of Experimental Examples 1 to 4, had significantly higher total phenolic compound contents than Comparative Examples 1 to 4 I could.

Test Example 5-2: Measurement of flavonoid content

The flavonoid contents of wild ginseng cells of Example 1 and Comparative Examples 1 to 4 were determined by modifying the David method. Namely, Naringin was prepared at a concentration of 0.0016 to 0.2 mg / ml and used as a standard reagent. Diethylene glycol and 1N NaOH were added to each sample at a concentration of 0.0016 to 0.2 mg / ml, Were used as experimental groups. The standard curve was then measured by UV spectrophotometer at 420 nm. 100 mg of lyophilized callus obtained by applying Coordinated media was taken in a 15 ml conical tube, and then 7 ml of 50% methanol (MeOH) was added, and the mixture was extracted with an ultrasonic extractor for 1 hour. I have used it. The extract was filtered and used as a test solution. 200 μl of the sample solution was mixed with 2 ml of diethyleneglycol, and 200 μl of 1 N NaOH was added. The mixture was allowed to stand in a water bath at 37 ° C. for 1 hour and then measured at 420 nm with a UV spectrophotometer. The results are shown in Table 6 below.

division Wavelength (nm) Transmittance Absorbance Concentration (mg / ml) naringin 0.002 (mg / ml) 420 99.36 0.003 0.002 naringin 0.02 (mg / ml) 420 89.30 0.049 0.023 naringin 0.2 (mg / ml) 420 43.14 0.365 0.199 Example 1 420 48.82 0.421 0.262 Comparative Example 1 420 32.15 0.304 0.181 Comparative Example 2 420 43.43 0.395 0.201 Comparative Example 3 420 35.23 0.366 0.192 Comparative Example 4 420 48.82 0.354 0.195

Referring to Table 5, it can be seen that Example 1, which showed excellent results in the antioxidative, anti-inflammatory and whitening effects of Experimental Examples 1 to 4, has a significantly higher total flavonoid content than Comparative Examples 1 to 4 .

Test Example 5-3: Measurement of ginsenoside content

The content of ginsenoside, which is a representative physiologically active substance of wild ginseng, was measured for Example 1 and Comparative Examples 1 to 4. The content of ginsenoside was measured by Shibata method. First, in order to measure the intracellular content, a sample was prepared as follows.

The extract of Example 1 is concentrated under reduced pressure and sterilized 25 is added to dissolve it. The extract was added to the aliquot, ether was added to remove the lipid layer, butanol was added, and the mixture was shaken and allowed to stand for one hour. This fractionation process was repeated twice, and the obtained butanol layer was concentrated under reduced pressure, and then dissolved in HPLC grade methyl alcohol, filtered using a 0.2 μm filter, and used as a test solution for HPLC analysis.

The results of the HPLC analysis of Example 1 are shown in FIG. 1, and the total content of jinzonide in Example 1 and Comparative Examples 1 to 4 is shown in Table 7 below.

Unit (mg / g) Example 1 Comparative Example One 2 3 4 Rg1 1.74 0.63 1.77 1.56 1.64 Re 2.23 1.49 1.57 2.05 2.03 Rf 0.47 0.33 0.36 0.43 0.47 Rh1 0.05 0.14 0.12 0.09 0.05 Rg2 0.5 0.28 0.28 0.37 0.5 Rb1 1.52 1.4 1.4 1.14 0.9 Rc 0.46 0.44 0.43 0.16 0.32 Rb2 0.49 0.35 0.38 0.35 0.49 Rd 0.17 0.2 0.27 0.19 0.17 F2 0.32 0.09 0.12 0.07 0.12 Rg3 (s) 0.05 0.04 0.02 0 0.01 Rg3 (r) 0.07 0.02 0.01 0.01 0.01 Rh2 0.18 0.2 0.27 0.17 0.18 PPD 0.13 0.11 0.07 0.13 0.13 C-K 0.06 0.01 0.03 0.02 0.01 PPT 0.13 0.18 0.21 0.22 0.13 Total 8.57 5.91 7.31 6.96 7.16

Referring to FIGS. 1 and 7, the contents of ginsenosides contained in the somatic embryos of the wild ginseng were higher than those of Comparative Examples 1 to 4.

Production Example 1: Preparation of Cosmetic (Lotion)

Using the ingredients listed in Table 8 below, lotions were prepared by conventional methods. 100 parts by weight of the wild ginseng cells of Example 1 were added to 2000 parts by weight of 70% (v / v) ethanol aqueous solution as an extract of the wild ginseng cell, and aged at room temperature for 1 week, followed by filtration through a filter paper to obtain a filtrate . The filtrate was concentrated under reduced pressure using a rotary vacuum evaporator (Eyela, Japan) to obtain an extract.

Ingredients Content (% by weight) Purified water 88.97 1,3-butylene glycol 3.0 glycerin 1.0 Sodium hyaluronate (1% aqueous solution) 2.0 antiseptic 0.01 Sodium citrate 0.01 Citric acid 0.01 Example 1 Extract 5.0 Sum 100

Production Example 2: Production of cosmetic (lotion)

Using the same ingredients as those of Example 1 above, the lotions were prepared using the ingredients listed in Table 9 below using conventional methods.

Ingredients Content (% by weight) Purified water 88.97 1,3-butylene glycol 2.0 glycerin 1.0 Carbomer 15.0 Triethanolamine 0.2 Stearic acid 3.0 Beads wax 0.5 Squalene 5.0 Glyceryl stearate 2.0 Cetyl alcohol 1.0 antiseptic 0.01 Example 1 Extract 5.0 Sum 100

Claims (3)

Inducing embryogenic callus from the cotyledon of Panax ginseng CA Meyer;
Culturing the embryogenic callus to induce somatic embryo cells; And
Culturing the somatic cell-cultured cells at a pH of 4.5 to 6.0 and a temperature of 10 to 25 DEG C,
Wherein the somatic embryo-cultured cells are subjected to ultrasonic stimulation and light stimulation during the culturing, thereby promoting the production of secondary metabolites of wild ginseng cells.
[3] The method according to claim 1, wherein the ultrasonic stimulation comprises treating the somatic cells with a frequency of 20 to 50 kHz and irradiating the somatic cells with a light having a wavelength of 380 to 430 nm. Methods for promoting metabolite production.
The method according to claim 1, wherein the average content of the phenolic compounds contained in the wild ginseng cells is 0.115-0.219 mg / g, the average content of flavonoids is 0.18-0.28 mg / g, and the average content of ginsenosides Is 5.85 to 8.65 mg / g.

Images