KR102105166B1 - A mass production method of Leontopodium coreanum Nakai in vitro culture and a composition for anti-oxidating, anti-inflammation and anti-wrinkle comprising extracts of Leontopodium coreanum Nakai - Google Patents

Abstract

The present invention relates to a composition for anti-oxidation, anti-inflammatory and anti-wrinkle that includes a mass production method using in-situ culture of som-legs and an extract of som-dari as an active ingredient, and in the present invention, the som-dari, a beneficial plant, is the same in a short period of time through in-flight culture Normal plants can be obtained by multiplying the sod leg mass and inducing rooting whenever necessary. In addition, the composition comprising the sodari extract of the present invention as an active ingredient has excellent total DPPH free radical scavenging activity and excellent NO production inhibition effect and elastase activity inhibition effect. Therefore, the cotton leg extract of the present invention has no cytotoxicity and skin side effects, and thus can be safely used in cosmetic and pharmaceutical food compositions.

Description

A mass production method of Leontopodium coreanum Nakai in vitro culture and a composition for anti-oxidating, anti-inflammation, including mass production method using in-flight culture of cotton leg and extract of cotton leg as an active ingredient and anti-wrinkle comprising extracts of Leontopodium coreanum Nakai}

The present invention relates to a composition for anti-oxidation, anti-inflammatory and anti-wrinkle comprising an in-flight culture method of a som leg ( Leontopodium coreanum Nakai) and an extract of a som leg as an active ingredient, and more specifically, a mass of a rare leg, a domestic rare species, by in-flight culture method. It relates to an antioxidant, anti-inflammatory and anti-wrinkle cosmetic and pharmaceutical composition comprising a method of producing and an extract of cotton leg as an active ingredient.

The sombridge ( Leontopodium coreanum Nakai) is a dicotyledonous plant that grows in deep mountain rocks. The origin is similar to that of the European Alps region, the alpine plant Edelweiss ( Leontopodium alpinum ), so it is often confused, but the som-dari is a Korean specialty species that grows in Halla and central north.

It is called a sombari because it grows right and is covered with white fluffy. The stems are gathered, and there are sacks with flowers and without flowers. The surface of the leaf is slightly fluffy and the back side is grayish white. The flowers bloom yellow in July-August, and 8-16 are gathered and run in a variety of inflorescences. The gun pieces are arranged in 3 rows, and the edges are black with white hairs on the back. The fruit is a fruit tree, ripening in October, about 1 mm long, long oval, with short hairs. The sombari has the effect of lowering heat, removing poison, relieving inflammation and stopping pain.

Recently, many studies have been conducted to develop functional foods using vegetable resources and to use them as cosmetic materials, but in the case of som legs, which are native plants in Korea, many studies have not been conducted as rare materials that are difficult to grow in Korea.

Therefore, in the field, there is a need for an in-flight culture method capable of mass-producing a useful plant resource, a soy leg.

On the other hand, human skin is composed of the epidermis, dermis, and connective tissue, including the stratum corneum, of which the dead layer is a dead cell layer formed through the differentiation of keratinocytes, the basal cells of the epidermis. It is composed and plays a role of protecting the human body from the influence of the external environment. The causes of aging in the skin have been studied in two major ways. One is "internal aging," which is due to changes in the function of cells, which are the structural units of the skin, with the increase in age, and the other, "external aging." It can be divided into external environments such as ultraviolet rays, pollution, and stress.

Cells of the skin, when oxidative stress is applied by free radicals occurring in a biochemical reaction that constantly occurs in order to supply the energy required in the human body to supply the energy required in the body, the cell components such as lipids, proteins, Carbohydrate and DNA oxidative damage and enzyme activity are changed to cause various diseases such as skin cancer and promote aging. In addition, the largest part of the causes of external aging is photoaging due to ultraviolet rays, which causes acute and chronic skin injury such as skin cancer through continuous exposure to UV.

Biosynthesis and degradation of collagen, one of the important components of the dermal matrix of the skin, is key to the inhibition of skin aging. The reactive oxygen species promote the expression of matrix metalloproteinases (MMPs) in skin fibroblasts, and collagenase by UV light. It promotes the synthesis of agents and causes skin aging.

The skin is a tissue that protects the organs in the body, and protects the body's moisture and acts as an essential barrier to protect the human body by preventing invaders from outside. Skin protects the body from pathogens against the external environment, regulates body temperature, and senses sensations.

Inflammatory response is a physiological response that protects the body from invasion and physical damage of the inducers of inflammation and induces an increase in immune-related cells and bioprotective substances, and these cells are inflamed by secreting various types of immune proteins and cytokines. It is responsible for the defense and treatment of the site. However, excessive aggregation of immune cells and production of immune mediators can cause damage to tissue cells, so proper regulation of the immune response is necessary. Factors that cause skin inflammation include physicochemical stimulation, allergens, ultraviolet rays, oxidative stress, pathogen infections, and secondary cytokines and chemokines. The general route of the skin inflammatory reaction is an immune response to protect the human body from external stimulation, and control of inflammation through suppression of the inflammatory response is a main target of treatment.

When a steroid-based anti-inflammatory drug is used as a conventional skin inflammation prevention or treatment method, the anti-inflammatory effect is excellent, but when used for a long time, there are problems such as thinning of the skin, discoloration of the skin, or prominent capillaries. In addition, there is a problem in that resistance is exhibited such that symptoms may worsen when the use is stopped.

In addition, when using non-steroidal anti-inflammatory drugs, the effect is temporary, there are problems such as osteoporosis, arteriosclerosis, carcinogenic potential. As such, when using a steroid-based or non-steroidal anti-inflammatory drug, there is a disadvantage as described above, and thus it is necessary to develop a composition that is safe from the natural product and has excellent anti-inflammatory effect.

In addition, elastin (elastin) fibers are cross-linked with collagen and are important skin components for wrinkles that are involved in skin elasticity. The deficiency and aggregation of elastin fibers, and a marked increase in the activity of the elastin degrading enzyme, elastase, have been found to be one of the factors causing skin wrinkles. Elastase is the only enzyme that can break down elastin, and its inhibition is known to fundamentally reduce skin wrinkle improvement.

Therefore, in the field of cosmetics, research and development of functional cosmetics having antioxidant, anti-inflammatory, and anti-wrinkle effects have been actively conducted, and in particular, research using natural substances has been continued to not cause toxicity or irritation to the skin.

In the present invention, the material is secured through the establishment of a systematic plant factory through cultivation of plant tissues for commercialization through continuous supply of small amounts of native or rare plant species in Korea, and the secondary metabolites are derived from the tissue culture method of simple culture. And it intends to secure technology that can massize it and use it for industrialization. In spite of the excellent active activity, in order to increase the businessability of the material, which is difficult to industrialize due to the difficulty in supply and supply of raw materials itself due to the lack of smooth supply of specific materials, the present invention disinfects seeds for tissue cultivation of selected plant materials And medium, hormone conditions, etc., to establish optimal culture conditions. As a way to produce this in large quantities and provide sustainable supply, we intend to induce callus through tissue culture technology and establish optimal plant production conditions using technology and plant plants capable of mass production.

In addition, in the present invention, as a result of continuing research to develop new natural substances having small side effects on the human body while having antioxidant, anti-inflammatory, and anti-wrinkle effects, the antioxidant, anti-inflammatory, and anti-wrinkle effects of the sombari extract without cytotoxicity The present invention was completed by confirming the fact that.

Therefore, the technical problem to be solved in the present invention is to provide an in-flight culture method of the sombari.

In addition, another technical problem to be solved in the present invention is to provide a composition for anti-oxidation, anti-inflammatory and anti-wrinkle containing a cotton ball extract.

In order to solve the above technical problem, the present invention provides a method for mass production of cotton wool by in-flight culture, comprising the following steps:

(S1) harvesting the leaves from the cotton leg, washing, sterilizing and then cutting;

(S2) Callus by adding indole acetic acid (IAA) and kinetin (NAA) or naphthalene-acetic acid (NAA) and 6-benzylaminopurine (BA) to MS medium to which sucrose and gelrite were added. ) Preparing an induction medium, and inducing callus generation by culturing the leaf sections sterilized in step (S1);

(S3) subculturing the callus generated in step (S2) to propagate the callus;

(S4) growing the callus by inoculating the callus propagated in step (S3) in the MS medium to which NAA and BA were added; And

(S5) Incubating the callus grown in the above step (S4) in B5 medium or MS medium to induce rooting, thereby preparing an in-vivo culture medium.

In order to solve the other technical problems described above, it provides a composition for antioxidant, anti-inflammatory and anti-wrinkle, characterized in that it comprises an extract of cotton sod as an active ingredient.

Preferably, in the present invention, the composition comprising the cotton ball extract may be a cosmetic composition or a pharmaceutical composition.

As used herein, the term "in vitro culture" is also referred to as in vitro culture, and refers to an in vitro culture for reproducing a substance drawn from an organism in vitro.

In general, the types of plant hormones used for in-plant culture of plants include BA (6-benzylaminopurine), kinetin (N-2-furanylmethyl-1H-purine-6-amine) and TDZ (Thiazuron-N-phenyl-N). Cytokinins, such as -1,2,3 thiadiazol-5ylurea); Auxins such as indole acetic acid (IAA), indole butyric acid (IBA), naphthalene-acetic acid (NAA) and 2,4-dichlorophenoxy-acetic acid (2,4-D); Gibberellins such as GA3. Plant hormones have different effective concentrations depending on the type of tissue applied, and it is often uncertain whether they are carried outside the synthesized site and exhibit the same function. Therefore, it is important to properly select the type and concentration of plant hormones according to the purpose, which may be difficult to predict and select without going through actual experiments.

The mass production method of the sombari by in-flight culture of the present invention includes (S1) washing, sterilizing, and cutting the leaves from the sombari; (S2) Callus by adding indole acetic acid (IAA) and kinetin (NAA) or naphthalene-acetic acid (NAA) and 6-benzylaminopurine (BA) to MS medium to which sucrose and gelrite were added. ) Preparing an induction medium, and inducing callus generation by culturing the leaf sections sterilized in step (S1); (S3) subculturing the callus generated in step (S2) to propagate the callus; (S4) growing the callus by inoculating the callus propagated in step (S3) in the MS medium to which NAA and BA were added; And (S5) incubating the callus grown in step (S4) in B5 medium or MS medium to induce rooting, thereby preparing an in-vivo culture medium.

In the in-flight culture method of the present invention, step (S1) is a step of washing and sterilizing and then cutting the cotton leg leaves, and the cotton leg leaves use young leaves, for example, by purchasing seedlings having a length of about 8 to 12 cm above the ground. Above 10 ° C., light-treated for 14 to 18 h, and young leaves before flowering were grown under dark treatment for 6 to 10 h.

The sterilization process is followed by primary sterilization in 70% ethanol after washing the soil on the leaves with running water, and 3 times with sterilized distilled water, followed by secondary sterilization in 1% sodium hypochlorite followed by distilled water 3 Wash repeatedly.

The cutting was used by cutting the sterilized leaf tissue to a size of 0.5 to 1.5 cm 2.

In the in-flight culture method of the present invention, step (S2) is a step of inducing callus development, and 2-3% sucrose and 0.3-0.5% gelite are added to MS medium to induce callus from sterilized leaf tissue. As a plant growth regulator, kinetin, NAA, BA, and IAA were used, and the pH of the medium was set within a range of 5.55 to 5.59, and cancer treatment was performed in a plant incubator at 20 to 30 ° C to induce callus.

In the in-flight culture method of the present invention, step (S3) is a step of propagating callus, and then inducing somatic cells by passage-incubation of the callus induced at least 4 times at 2 week intervals, followed by passage of culture at 4 week intervals after 8 weeks. do.

The "subculture" is a method of continually culturing the stem of a cell by transferring it to a new culture medium for cell proliferation, which prevents the cell from stopping proliferation in a limited culture medium and allows the cell to proliferate. It is to provide space.

In the in-flight culture method of the present invention, step (S4) is a step of growing the propagated callus, and the calla is added to the MS basic suspension culture (pH 5-6) to which NAA (2 mg / L) and BA (1 mg / L) are added. It was inoculated, and sealed and then incubated in a stirrer. At this time, the culture room temperature was adjusted to 20 ± 3 ° C., and the primary medium was cultured for 1 to 2 weeks by adjusting the photoperiod with light treatment for 12 to 16 h and dark treatment for 8 to 12 h, and then the suspension medium was added under the same culture conditions. The first increase was carried out, and the suspension medium was subjected to the second increase in accordance with the callus growth rate, and a total of 4-6 weeks was performed.

In the in-situ culture method of the present invention, step (S5) is a step of generating an in-situ culture medium and incubating the grown callus in B5 medium or MS medium for about 2 months to induce rooting to obtain an in-situ culture medium.

According to one embodiment of the present invention, the mass production method of the sombari by in-flight culture of the present invention may further include the step of purifying the in-flight culture obtained in the step (S5).

The "acclimation" refers to the process of adapting a plant by gradually adjusting the growth environment (temperature, humidity, light conditions) for a period of time, because it may not be able to adapt if a young plant that has been differentiated and grown by in-flight culture is grown in an external environment. it means.

In the purifying process, the in-flight culture medium is washed with sterile water to remove the medium component, and the in-flight culture medium is transferred to a port containing sterilized culture soil (vermiculite), covered with a plastic bag, at 80-90% humidity and 28-32 ° C 2 It can be performed by growing for ~ 5 weeks (eg, 4 weeks). The growth may be performed by replenishing nutrients and moisture using 1/2 MS medium.

The purified plants can be transferred to a greenhouse, which is an external environment, and grown. Specifically, the plant can be transferred to a pot filled with sterile culture soil to grow under shade conditions, and then transferred to a large pot filled with horticultural soil as the plant grows.

According to one embodiment of the present invention, there is provided a sombari prepared by a method of mass-proliferation of a sombari by in-flight culture.

In the present invention, a plant factory for stable supply and demand of natural plant materials was designed. Plant factories are systems that produce plants semi-closed in a closed or semi-closed space that regulates the internal environment, and artificially control environmental conditions such as light, temperature, humidity, carbon dioxide concentration, and culture medium to relate to seasons or places. It is a system that continuously produces plants without a plant. Some plant factories also produce useful metabolites by cultured cells or apply genetic engineering to produce foreign substances in plants. In other words, it can be said that the plant factory is an environment-preserving production system aimed at supplying safe food and supplying cutting materials to the anniversary.

The present invention provides a composition for antioxidant, anti-inflammatory, and anti-wrinkle, characterized in that it contains an extract of cotton sod as an active ingredient.

In the present invention, the "active ingredient" refers to a component that can exhibit the desired activity alone or itself can exhibit activity with an inactive carrier.

The term "extract" of the present invention refers to a formulation in which a crude drug is squeezed into an appropriate leach solution and concentrated by evaporating the leach solution, but is not limited thereto, an extract obtained by an extraction treatment, a dilution or concentrate of the extract, or an extract It may be a dried product obtained by drying, a crude product or a purified product thereof.

The sombari extract of the present invention can be prepared using general extraction methods, separation and purification methods known in the art. The extraction method may include hot water extraction, hot water extraction, cold needle extraction, reflux cooling extraction, or ultrasonic extraction, but is not limited thereto.

According to the embodiment of the present invention, the cotton leg extract may be extracted by using a solvent selected from the group consisting of an extraction solvent, for example, water, anhydrous or hydrous lower alcohol having 1 to 3 carbon atoms, and a mixed solvent thereof, as an extraction solvent, , The amount of the extraction solvent may be 2 to 30 weight times of the crude drug weight, preferably 10 to 20 weight times.

In one specific embodiment of the present invention, the cotton ball extract may be extracted by immersing the cotton ball in ethanol, and then filtered with filter paper to obtain a cotton ball extract.

In the present invention, any part of the leaves, stems, and roots of the sombari may be used.

In the composition of the present invention, the cotton ball extract may contain 0.005 to 50% by weight, more preferably 0.01 to 30% by weight, and most preferably 0.1 to 10% by weight based on the total weight of the composition. At this time, if the content of the extract of cotton leg is less than 0.005% by weight, it is impossible to obtain the antioxidant effect, which is the objective effect of the present invention, and if it exceeds 50% by weight, the effect is not proportional to the increase in content, so it may be inefficient. There is a problem that the stability of the.

The composition containing the cotton leg extract of the present invention exhibits antioxidant, anti-inflammatory and anti-wrinkle effects, and has little cytotoxicity as a natural substance.

According to one embodiment of the present invention, there is provided a cosmetic composition comprising a cotton ball extract as an active ingredient.

In the cosmetic composition according to one embodiment of the present invention, in addition to the sodari extract as an active ingredient, ingredients conventionally added to cosmetic compositions, such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavors, common adjuvants, and carriers Can be further added.

The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleaning , May be formulated as an oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it can be prepared in the form of a nutrition cream, astringent lotion, soft lotion, lotion, essence, nutrition gel or massage cream.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, trakant gum, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide are used as carrier components. Can be.

When the formulation of the present invention is a powder or a spray, toss, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder can be used as a carrier component, and in the case of a spray, additionally chlorofluorohydrocarbon, propane / Propellant such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

When the formulation of the present invention is a suspension, liquid diluents such as water, ethanol or propylene glycol as carrier components, ethoxylated isostearyl alcohol, suspensions such as polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, microcrystals Sex cellulose, aluminum metahydroxide, bentonite, agar or trakant gum and the like can be used.

When the formulation of the present invention is a surfactant-containing cleansing, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivatives, methyltaurate, sarcosinate, fatty acid amide as a carrier component Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

The pharmaceutical composition according to one embodiment of the present invention includes a pharmaceutically acceptable carrier in addition to the cotton ball extract. The pharmaceutically acceptable carrier included in the pharmaceutical composition of the present invention is commonly used in formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, but is not limited thereto It does not work. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. 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 be administered orally or parenterally, preferably parenteral administration, more preferably applied in a topical application mode by application.

Suitable dosages of the pharmaceutical compositions of the invention are variously prescribed by factors such as formulation method, mode of administration, patient's age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion, and response sensitivity. Can be. The dosage of the pharmaceutical composition of the present invention is in the range of 0.001-100 mg / kg on an adult basis. In addition, in the case of an external preparation, it is recommended to apply for 1 to 5 times a day in an amount of 1.0 to 3.0 ml on an adult basis and continue for at least 1 month. However, the dosage is not intended to limit the scope of the present invention.

The pharmaceutical composition of the present invention is prepared in a unit dose form or multi-dose by formulating using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily carried out by those skilled in the art. It can be prepared by incorporating into a container. In this case, the formulation may be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or may be in the form of an exir, powder, granule, tablet or capsule, and may further include a dispersant or stabilizer.

On the other hand, the cotton leg extract of the present invention is a natural substance, which is harmless to the human body and has little toxicity and side effects, so it can be used safely even for long periods of use. In particular, it can be safely applied to cosmetics and pharmaceutical compositions as described above.

As described above, in the present invention, a normal plant can be obtained by inducing a mass of the same sombreal object in a short period of time through in-flight culture, which is a beneficial plant, and inducing rooting whenever necessary. In addition, the composition comprising the sodari extract of the present invention as an active ingredient has excellent total DPPH free radical scavenging activity and excellent NO production inhibition effect and elastase activity inhibition effect. Therefore, the cotton leg extract of the present invention has no cytotoxicity and skin side effects, and thus can be safely used in cosmetic and pharmaceutical food compositions.

1 is a photograph of the sombrero young leaves to be used in the experiment.
2 is a photograph of callus derived from a sombari leaf.
Figure 3 is a photograph showing the growth pattern of the in-plane culture in dentate soybeans in B5 and MS medium.
Figure 4 shows the results of spectral analysis of the fraction of the cotton leg extract.
Figure 5 shows the results of 1H-NMR and 13C-NMR spectral analysis of compounds [VFE-6-4-3, 46mg, Ve / Vt 0.379 to 0.506, ODS TLC Rf 0.679, acetone-H2O = 5: 1] in the sombari fraction. It is shown.
Figure 6 is a graph showing the results of the cytotoxicity test of the sombari extract.
7 is a graph showing the results of DPPH electron donating ability according to the concentration of the sodari extract.
8 is a graph showing the degree of inhibition of NO production according to the concentration of the sodari extract.
9 is a graph showing the inhibitory activity of elastase according to the concentration of the sodari extract.

Hereinafter, examples and the like will be described in detail to help understanding of the present invention. However, the embodiments according to the present invention can be modified in many different forms, and the scope of the present invention should not be construed as being limited to the following examples. The embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

<Example 1> Plant factory design for stable supply and demand of natural plant materials

(1) Experimental materials

The som legs used in the experiment were purchased with seedlings having a length of about 10 cm above the ground, light-treated for 10 h or more, 16 h, and grown under dark treatment conditions for 8 h to use young leaves before flowering. 1 is a photograph of the sombrero young leaves to be used in the experiment.

(2) Tissue sterilization and section denture

After washing the soil on the leaves with running water, it was first sterilized in 70% ethanol for 1 minute, washed three times with sterile distilled water, and then sterilized in 1% sodium hypochlorite for 20 minutes, followed by distilled water Washed repeatedly with 3 times. Thereafter, the sterilized leaf tissue was cut into 1 cm 2 size, and then placed on the medium for culture of callus according to the composition (mg / L) of the medium plant growth regulator as shown in Table 1 below.

IAA
(Indole acetic acid) Kinetic
(Kinetin) NAA
(Naphthalene-acetic acid) BA
(6-benzylaminopurine) M1 0.1 0.2 - - M2 0.2 0.3 - - M3 0.3 0.4 - - M4 - - 0.5 0.5 M5 - - 1.0 2.0 M6 - - 2.0 1.0

(3) Callus division induction and suspension culture

2-3% sucrose and 0.4% gelite were added to MS (Murashige and Skoog 1962, Duchefa, Netherlands) medium to induce callus from sterile leaf tissue, and plant growth as shown in Table 1 above. Different concentrations of kinetin, NAA, BA, and IAA were used by treating the modulators differently, and the pH of the medium was set within the range of 5.55 to 5.59. The callus was induced by cancer treatment in a plant incubator at 25 ° C. The induced callus was passaged at least 4 times at 2 week intervals to propagate the cells, and after 8 weeks, passage was performed at 4 week intervals to induce somatic cells. Suspension culture was inoculated with 1 g of callus in a 500 mL flask containing 250 mL of MS basic culture solution (pH 5.5) containing NAA (2 mg / L) and BA (1 mg / L) selected as the optimal medium, and sterilized. After sealing with an aluminum foil, it was incubated at a speed of 120 rpm in a stirrer. The culture room temperature was adjusted to 20 ± 3 ° C., and the primary medium was cultured for 1 to 2 weeks by adjusting the photoperiod by light treatment for 14 h and dark treatment for 10 h, and then the primary suspension was increased to 250 mL under the same culture conditions. , In accordance with the growth rate of callus, 250 mL of suspension medium was subjected to the second increment, and was performed for a total of 4-6 weeks.

To measure the growth rate of callus according to the composition of the medium, the suspension-cultured cells are filtered with Whatmam filter paper and separated from the culture medium, and then dried cells are dried at 65 ° C for 3 days to measure the dry weight value and Comparison was made and an experiment was conducted to confirm the effect of Elicitor treatment on the growth of the individual. Specifically, when inoculating callus into 250 mL suspension culture, MeJA (100 μM) was treated with 1%, and then cultured under the same conditions as the existing suspension culture conditions, and the same amount of effluent was applied in the first and second increments. Did. 2 is a photograph of callus derived from a sombari leaf.

(4) Production of in-flight cultures for plant factories

For industrial mass production, growth experiments were carried out with different media compositions to search for optimal growth conditions from various conditions. This confirms that the amount of growth may vary depending on the type of nutrient supply required for plant growth, and the optimum growth condition was established by measuring the growth amount of the in-flight culture medium through various media conditions.

In this experiment, Gamborg B5 Medium, CHU (N6) Medium, Murashige & Skoog Medium (MS), Woody Plant Medium (WPM), and Schenk & Hidebrandt Medium (SH) were used to grow in-flight cultures, respectively. The humidity conditions were kept the same to determine the optimum nutritional components needed for in-flight culture.

The composition of B5 and MS medium used in this experiment is as shown in Table 2 below, and each medium was prepared by dividing it into original medium, 2/3, 1/2, and 1/4, respectively, and in-flight culture indented on each medium. Was grown under in-flight conditions for 2 months, and then the growth amount was confirmed.

Elements Gamborg B5 Medium (mg / L) Murashige & Skoog Medium (mg / L) CoCl ₂ · 6H ₂ O 0.025 0.025 CuSO ₄ · 5H ₂ O 0.025 0.025 FeNaEDTA 36.70 36.70 H ₃ BO ₃ 3.00 6.20 KI 0.75 0.83 MnSO ₄ · H ₂ O 10.00 16.90 Na ₂ MoO ₄ 2H ₂ O 0.25 0.25 ZnSO ₄ · 7H ₂ O 2.00 8.60 CaCl ₂ 113.23 332.02 KNO ₃ 2500.00 170.00 MgSO ₄ 121.56 1900.00 NaH ₂ PO ₄ 130.44 180.54 (NH ₄ ) ₂ SO ₄ 134.00 1650.00

As a result, in-flight culture failed in most of the test groups, but smooth growth was confirmed in the group grown in B5 and MS medium. Figure 3 is a photograph showing the growth pattern of the in-plane culture in dentate soybeans in B5 and MS medium.

Through these experiments, in-flight planting conditions were established for the sod leg plants, which is significant in that it is possible to produce rare materials that are difficult to grow domestically throughout the year.

<Example 2> Preparation of cotton leg extract

The cotton leg was extracted by dipping 2 kg in 80% EtOH (50 L) at room temperature for one week.

The extract obtained by repeating extraction twice in the same manner was filtered and concentrated under reduced pressure to obtain 260.42 g of ethanol extract, dissolved in water, and then partitioned into n-hexane, ethyl acetate (EtOAc), and butyl alcohol (n-BuOH) according to the polarity. Four fractions were extracted (Hexane 42.34g, EtOAc 67.56g, BuOH 50.12g, Water 86.94g).

Silica gel column chromatography from EtOAc fraction (67.56 g) (Φ 15 cm X 23 cm, n-Hexane-EtOAc = 4: 1 → 1: 1 → CHCl3-MeOH = 50: 1 → 35: 1 → 30: 1 → 13 : 1 → 8: 1 → 5: 1) was performed with a gradient, and each fraction was identified by SiO2 TLC, similar parts were collected together, and concentrated to obtain 12 fractions (VFE-1 to VFE-12). Among them, the VFE-6 fraction (7.1 g, Ve / Vt 0.547 to 0.613) was subjected to ODS column chromatography (Φ 7 cm X 12 cm, acetone-H ₂ O = 1: 1) to perform 9 fractions (VFE- 6-1 to VFE-6-9), of which VFE-6-4 fraction (670 mg, Ve / Vt 0.551 to 0.674) was obtained by SiO ₂ column chromatography (Φ 3 cm X 8 cm, CHC _l3) -MeOH-H2O = 13: 3: 1) to give 8 fractions (VFE-6-4-1 ~ VFE-6-4-8).

Figure 4 shows the results of spectral analysis of the fraction of the cotton leg extract.

< Example  3> Cotton leg Fractions  NMR analysis and structure identification

Among the eight fractions separated in Example 2, the compound [VFE-6-4-3, 46mg, Ve / Vt 0.379 ~ 0.506, ODS TLC Rf 0.679, acetone-H ₂ O = 5: 1] was separated and separated. The activity of the compound was confirmed, and the compound having activity was dissolved in MeOH (CD ₃ OD) to perform NMR analysis, and ¹ H-NMR and ¹³ C-NMR were measured by a 300 MHz FT-NMR spectrum.

5 is a ¹ H-NMR and ¹³ C-NMR spectrum analysis of the compound [VFE-6-4-3, 46mg, Ve / Vt 0.379 ~ 0.506, ODS TLC Rf 0.679, acetone-H2O = 5: 1] in the sombari fraction The results are shown.

As can be seen here, the compound (Rutin) (yellow crystal) confirmed the presence of ketone (1656 cm ^-1 ) and hydroxyl group (3421 cm ^-1 ) in the IR spectrum. Molecular weight of m / z 609 [MH] In the EI / MS ^- is the observed molecular weight was determined to 610. As a result of NMR analysis of the separated compound, 3 olefin methine protons (δH 7.67, 1H, d, J = 2.01Hz; 7.64, 1H, dd, J = in the 1H-NMR (300MHz, CD3OD, δH) spectrum 8.43,2.01Hz; 6.88, 1H, d, J = 8.43Hz) signal was observed to predict the presence of 1,2,4-3 substituted benzene rings, and two olefin methine protons (δH 6.37, 1H, d, It was expected that 1,2,3,5-4 substituted benzene rings were present through J = 2.01Hz; 6.19,1H, d, J = 2.01Hz) signals.

A number of proton signals were observed by observing a number of proton signals in the hydroxyl group, thereby predicting a compound in which sugar is bound to the flavonoid skeleton. In addition, a total of 27 carbon signals were observed in the ¹³ C-NMR (100 MHz, CD3OD, δc) spectrum.

One carbonyl carbon [δC 179.37 (C-4)] signal was observed in the low-length region, and seven oxidized olefin quaternary carbons [δC 166.04 (C-7), 162.92 (C-5), in the olefin region. 159.29 (C-9), 158.47 (C-2), 149.78 (C-4 '), 145.79 (C-3'), 135.61 (C-3)] signals, and two olefin quaternary carbons [δC 123.08 ( C-1 '), 105.57 (C-10)] signal, 5 olefin quaternary carbons [δC 123.55 (C-6'), 116.03 (C-5 '), 117.69 (C-2'), 102.40 (C -6), 99.95 (C-8)] signal was observed.

10 oxidized methine carbons in the hydroxyl region [δC 104.74 (C-1 ″), 102.40 (C-6), 78.15 (C-5 ″), 77.16 (C-2 ″), 75.71 (C-4 ″), 73.91 (C-6 ″), 72.21 (C-3 ″ ′), 72.68 (C-2 ″ ′), 71.35 (C-4 ″), 69.69 (C-5 ″ ′)] signals and 1 oxidized styrene Carbon [δC 68.53 (C-6 ″)] signal was observed, and one methyl carbon [δC 17.88 (C-6 ″ ′)] signal was observed in the high magnetic field region. One North was expected.

In a 2D technique, the gHMBC experiment, the oxidized methine proton (δH 5.11, 1H, d J = 7.32Hz) signal at position 1 (H-1 ″) of glucose and the oxidized olefin quaternary carbon [δC 135.61 (C-3) )] As the cross peak of the signal was confirmed, the binding position of glucose was confirmed, and the oxidized methine proton (δH 4.52, 1H, s) signal at glucose 1 position (H-1 ″ ′) and glucose carbon 6 [δC 68.53 (C-6 ″)] As the cross peak of the signal was confirmed, the binding position of rhamnose was confirmed.

By synthesizing this, the compound separated from the leaf / stem was structurally identified by a routine (quarcetin-3-O-rutinoside) in which glucose and rhamnose were combined at the 3rd position of quassetin.

<Example 4> Toxicity evaluation of in-plane culture medium extract (MTT)

MTT analysis is an experiment to determine the cytotoxicity and survival rate.When MTT tetrazolium is treated with cells, MTT is reduced by living cells by absorbance measurement by dehydrogenase in living cells to reduce MTT formazan crystals. Known amount can be quantitatively evaluated.

The dermal papilla cells (DP cells) were treated with the extracts of the sombari in vitro cultures prepared in Example 2 by concentration and cultured for 24 h cells. The MTT reagent was treated and reacted in a 37 ° C. incubator for 4 h, and DMSO was reacted for 30 min, and absorbance was measured at 540 nm.

Figure 6 is a graph showing the results of the cytotoxicity test of the sombari extract. As can be seen here, cytotoxicity was not observed at all concentrations treated with the cotton leg extract.

<Example 5> Antioxidant effect of in-plane culture medium extract (DPPH)

Since oxidation reactions caused by free radicals in the body cause aging and cancer of cells, inhibition of free radical production may eventually be associated with antioxidant and anti-aging in the body. The antioxidant effect mainly uses the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical. DPPH is a method of indirectly testing how much the oxidative reaction that adversely affects the skin can be suppressed, and how much the sample reduces free radicals. The DPPH radical, which has a dark purple color, turns into a pale yellow color when it encounters an antioxidant and reacts, and measures the absorbance at a wavelength of 517 nm to evaluate the antioxidant capacity. The higher the antioxidant ability, the lighter the color and the lower the absorbance.

After adding 0.2 mM DPPH solution to a 96-well plate, it was reacted with ascorbic acid, a positive control. At this time, water was used as the solvent, and the in-plane culture extract was treated by%. After reacting for 30 min, absorbance was measured at 517 nm.

7 is a graph showing the results of DPPH electron donating ability according to the concentration of the sodari extract. As can be seen here, free radical scavenging activity was high at a concentration of 1% or more of the in-plane culture extract.

<Example 6> Anti-inflammatory effect of in-plane culture medium extract (NO assay)

Lipopolysaccharide (LPS) is a substance that causes an inflammatory reaction. Therefore, Griess reagent was used to measure NO production inhibition of the in-plane culture extract in Raw 264.7 cells activated with 1 mg / ml LPS. After 1 h of extract pretreatment, LPS treatment was performed to culture the cells for 24 h, followed by 50 μl of Griess reagent cell culture supernatant and Griess reagent (1% sulfanilamide in 5% phosphoric acid, 1% naphthylamide in H ₂ O) 100 μl The mixture was reacted for 10 min in a 96-well plate, and absorbance was measured at 540 nm. The NO concentration was diluted with sodium nitrate, and absorbance was measured to obtain a standard curve.

8 is a graph showing the degree of inhibition of NO production according to the concentration of the sodari extract. As can be seen here, NO inhibitory activity was observed from a concentration of 1% or more in the in-plane culture medium.

<Example 7> Elastase inhibitory activity of cotton in-plane culture extract

Elastase (Elastase) is involved in the decomposition of elastin, a matrix protein that maintains skin structure and elasticity in the dermis, and is an enzyme that can degrade collagen, causing wrinkles and reducing the elasticity of the skin. As age increases, elastase activity increases, skin wrinkles are generated and skin wrinkles can be prevented by inhibiting elastase activity.

After adding 100 mM Tris-HCl buffer (pH 8.0) to the 96-well plate, the in-plane culture extract was added by concentration. 2mM N-succinyl- (Ala) -3-p-nitroanilide was dispensed into each well, mixed with 0.2 unit of elastase enzyme prepared, and then reacted for 30 min in a 37 ° C incubator. After stopping for 5 min on ice to stop the reaction, absorbance was measured at a wavelength of 410 nm.

9 is a graph showing the inhibitory activity of elastase according to the concentration of the sodari extract. As can be seen here, it was shown that the elastase activity was significantly inhibited at a concentration of 0.125% or more of the cotton leg extract.

From the above results, the sombari in vitro culture extract does not show cytotoxicity, has high DPPH free radical scavenging ability, has NO inhibitory activity, and inhibits elastase activity, which is shown to be highly involved in antioxidant, anti-inflammatory and wrinkle improvement. .

Claims (8)

(S1) harvesting the leaves from the cotton leg, washing, sterilizing and then cutting;
(S2) Callus by adding Indole acetic acid (IAA) and Kinetic (Kinetin) or naphthalene-acetic acid (NAA) and 6-benzylaminopurine (BA) to MS medium with sucrose and gelrite added ) Preparing an induction medium, and inducing callus generation by culturing the leaf sections sterilized in step (S1);
(S3) subculturing the callus generated in step (S2) to propagate the callus;
(S4) growing the callus by inoculating the callus propagated in step (S3) in the MS medium to which NAA and BA were added; And
(S5) culturing the callus grown in step (S4) in B5 medium or MS medium to induce rooting to prepare an in-vivo culture medium;
Including,
In step (S1), seedlings having 8-12 cm in length above the soybean leaf are light-treated for 10 to 18 ° C., for 14 to 18 h, and young leaves before flowering while growing under dark treatment conditions for 6 to 10 h. Mass production method of the sombari by in-flight culture, characterized in that. delete According to claim 1,
In step (S1), the sterilization process is first washed with 70% ethanol after washing the soil on the leaves, and 3 times with sterile distilled water, followed by 2% with 1% sodium hypochlorite. After sterilization, a method of mass production of sombari by in-flight culture, characterized in that it is repeatedly washed three times with distilled water. According to claim 1,
Method of mass production of cotton wool by in-flight culture, characterized in that the culture in the step (S2) is a cancer treatment in a plant incubator of 20 to 30 ° C in a culture medium of pH 5.55 to 5.59. According to claim 1,
In step (S4), the growth is inoculated with callus on a suspension medium having a pH of 5 to 6, sealed to adjust the culture room temperature to 20 ± 3 ° C., light treatment for 12 to 16 h, and dark treatment with cancer treatment for 8 to 12 h A method for mass production of cotton wool by in-flight culture, characterized in that the suspension medium is first and secondly increased under the same culture conditions after primary cultivation for 1 to 2 weeks by adjusting the phase. According to claim 1,
A method for mass production of cotton wool by in-flight culture, further comprising the step of purifying the in-flight culture medium obtained in the step (S5). delete delete

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