KR102319658B1 - Method of producing callus of rock samphire for preparing rock samphire derived secondary metabolites - Google Patents

Abstract

The present invention relates to a manufacturing method of a rock samphire callus for manufacturing a secondary metabolite derived from rock samphire. More specifically, for mass producing through the increase of a phenolic compound which is the secondary metabolite derived from the rock samphire, the present invention relates to a manufacturing method of a rock samphire callus for increasing chlorogenic acid, caffeic acid, and ferulic acid, which are well-known intracellular active ingredients. The manufacturing method of the rock samphire callus comprises: a first step of inducing a plant; a second step of inducing a callus; a third step of selecting and increasing an excellent callus; and a fourth step of suspension cultivating.

Description

Manufacturing method of rock sampire callus for producing secondary metabolites derived from loxampire

The present invention relates to a method for producing a loxampire callus for producing a secondary metabolite derived from loxampire, and more particularly, to a method for mass production by increasing a phenolic compound that is a secondary metabolite derived from loxampire. It relates to a method for preparing a Roxampire callus for increasing chlorogenic acid, caffeic acid and ferulic acid, which are well known as my active ingredients.

Rock samphire ( Crithmum maritimum ) is a plant of the family Hepatica, which is mainly found in crevices of coasts, rocks, and cliffs in Europe. am. Halophytes are known to have strong resistance to high salinity, strong UV rays and wind due to their growth characteristics compared to other land plants. give the cause

Plants produce secondary metabolites as intermediates or products of interaction and defense metabolism between plants or with the external environment and microorganisms in addition to primary metabolites such as carbohydrates, lipids, and proteins necessary for life maintenance and growth. , these substances mainly protect the plant itself by producing and accumulating more when the plant is placed in an extreme environment. Among them, there are many expensive substances used as medicines, dyes, reagents, and industrial raw materials. In particular, plant-derived natural substances as pharmaceuticals have the advantages of mild and continuous action compared to synthetic substances, and have fewer side effects due to their high biodegradability.

In the case of loxampire, chlorogenic acid, a major secondary metabolite produced in the leaves of plants, is a phenolic compound composed of an ester bond of caffeic acid and quinic acid. It is known to have therapeutic effects on metabolic syndrome (anti-lipidosis, anti-diabetic and anti-obesity) (Santana-Gㅱlvez J et al. Molecules 2017; 22(3), 358). In the cosmetic composition, loxampire extract is known to be effective in inhibiting percutaneous moisture evaporation, improving wrinkles, and improving skin elasticity.

Unlike land plants, loxampire, which is attracting attention as a useful and high value-added plant, is a halophyte that grows in coastal areas by the sea. Since the content of the ingredients is not constant, it is difficult to secure a large amount. Therefore, there is a need for commercial production and use of secondary metabolites showing excellent physiologically active effects through overcoming these problems using the development of plant cell/tissue culture systems.

Plant cell and tissue culture technology is one of the biotechnology technologies that suggests that plant cell tissues can form whole cells and regenerate plants based on the totipotency of plants. Utilizing plant cell and tissue culture technology, grow disease-free plants from which microorganisms (such as fungi, bacteria, and viruses) and pests (insects and nematodes, etc.) propagating in higher plants are removed, and inducing and inducing undifferentiated stem cells, callus, therefrom. By selecting and securing excellent species and improving productivity and quality through optimal growth environment conditions, it may be possible to secure a large amount of plant-derived natural active materials.

Existing research and development using Roxampire mainly relates to cosmetic compositions and methods for manufacturing the same using freeze-dried products of callus culture or cells. The use of a freeze-dried product of plant cells is described, and in Korean Patent Application Laid-Open No. 10-2020-0031120, a 'cosmetic or pharmaceutical composition for promoting hair growth, including a freeze-dried product of dedifferentiated loxampire cells' is described. And, Korean Patent No. 10-2100646 describes a 'cosmetic composition comprising stem cell stem cells, which are callus cultures of Loxampire'. However, studies and analyzes in terms of cultivation physiology and biotechnology to increase the content of chlorogenic acid, an active ingredient of loxampire and their derivatives, have hardly been conducted.

Under this technical background, the inventors of the present application have researched and mass-produced callus culture and mass production of high value-added loxampire with increased content of chlorogenic acid and their derivatives (caffeic acid and ferulic acid), which are natural active materials based on plant cells and tissue culture. developed and completed the present invention.

1. Korea Laid-Open Patent Publication No. 10-2006-0118571 (2006.11.23) 2. Korean Patent Laid-Open Publication No. 10-2020-0031120 (2020.03.23) 3. Korea Patent Publication No. 10-2100646 (2020.04.08)

The first technical problem to be solved by the present invention is the production of loxampire callus for mass production of loxampire-derived secondary metabolites with excellent antioxidant, anti-inflammatory and antibacterial effects for the purpose of mass production of natural active materials based on plant cell and tissue culture to provide a method.

A second technical problem to be solved by the present invention is to provide a cosmetic composition comprising, as an active ingredient, a loxampire callus prepared by the above method.

In order to solve the first technical problem described above, the present invention provides a first step of sterilizing and germinating the loxampire seeds and then inducing the plants; a second step of inducing callus by culturing the plant tissue or an explant thereof in a medium to which auxin and cytokinin are added; A third step of selecting and proliferating excellent callus that can be preserved as a uniform culture with little variation among the callus; and a fourth step of suspending and culturing the proliferated callus.

The secondary metabolite may include, for example, a phenol-based compound as a nonvolatile secondary metabolite. Specifically, the secondary metabolite may be at least one selected from the group consisting of chlorogenic acid, caffeic acid, and ferulic acid.

The auxin is at least one selected from the group consisting of NAA (1-naphthaleneacetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid), and the cytokinin is 6-BA (6-benzylaminopurine) and kinetin (kinetin). ) may be one or more selected from the group consisting of.

The disinfection can be performed by immersing the Roxampire seeds in a solution containing 2-5% sodium chlorite (Soduim hypochlorite, NaOCl) and 0.01-0.02% surfactant.

The leaf and petiole of the plant, or explants thereof, are dented in a medium to which one or more combinations of auxins and cytokinins selected from the group consisting of:

A: Kinetin 0.5 mg/l, NAA 1.5 mg/l and 2,4-D 0.5 mg/l;

B: 6-BA 1 mg/l, NAA 1.5 mg/l and 2,4-D 0.5 mg/l; and

C: 6-BA 1 mg/l and NAA 1.5 mg/l.

The suspension culture may be performed by culturing the grown callus in a medium containing 1 mg/l of 6-BA and 1.5 mg/l of NAA.

In order to solve the second technical problem described above, the present invention provides a cosmetic composition comprising, as an active ingredient, loxampire callus prepared by the above-described method.

According to the present invention, it is possible to establish culturing conditions for roxampire in which the content of chlorogenic acid and its derivatives (caffeic acid and ferulic acid) having excellent antioxidant, anti-inflammatory and antibacterial effects is significantly improved, thereby depleting plant resources due to infinite growth and production and environmental protection. It is possible to supply chlorogenic acid and its derivatives (caffeic acid and ferulic acid) without destruction. It can be usefully used as a cosmetic composition.

The technical effects of the present invention are not limited to those mentioned above, and other technical effects not mentioned will be clearly understood by those skilled in the art from the following description.

1 shows the cell induction process from the leaves (leaf, L) and petiole (P) of the Roxampire plant grown through germination after sterilizing the seeds for in-flight introduction, selection of good stock, and suspension culture of callus. shows the process of
Figure 2 shows the flavonoid and total phenol content of non-volatile secondary metabolites of leaf extract (LE) and callus extract (CE) of loxampire.
3 is an HPLC analysis result of A) callus extract (CE) and B) leaf extract (LE) of Roxampire, and the content of 5 types of phenolic compounds obtained through this and the increase rate in callus compared to leaves is shown in C).
4 is an HPLC analysis result of a culture extract according to light conditions in culturing a callus extract (CE) of Roxampire, and shows the content and increase rate of five types of phenolic compounds obtained through this comparison.

While the present invention is susceptible to various modifications and variations, specific embodiments thereof are illustrated and shown in the drawings and will be described in detail hereinafter. However, it is not intended to limit the invention to the particular form disclosed, but rather the invention includes all modifications, equivalents and substitutions consistent with the spirit of the invention as defined by the claims.

Although the terms first, second, etc. may be used to describe various elements, components, regions, layers and/or regions, such elements, components, regions, layers and/or regions are not It will be understood that they should not be limited by these terms.

The inventors of the present application relate to the increase of phenolic compounds, which are representative secondary metabolites of the plant system, and in particular, chlorogenic acid, caffeic acid, and ferulic acid, which are well known as intracellular active ingredients ) was confirmed to increase the loxampire callus culture method.

Specifically, a culture method was confirmed to increase the content of phenolic compounds chlorogenic acid, caffeic acid and ferulic acid by culturing callus derived from Roxampire leaves and petioles in suspension. A method for producing a callus capable of mass-producing chlorogenic acid, caffeic acid and ferulic acid and the callus prepared by the method were invented.

Based on this, the present invention in one aspect, a first step of sterilizing the locksampire seed, germinating and then inducing the plant; a second step of inducing callus by culturing the plant tissue or an explant thereof in a medium to which auxin and cytokinin are added; A third step of selecting and proliferating excellent callus that can be preserved as a uniform culture with little variation among the callus; and a fourth step of culturing the proliferated callus in suspension.

The secondary metabolite may include, for example, a phenol-based compound as a nonvolatile secondary metabolite. Specifically, the secondary metabolite may be at least one selected from the group consisting of chlorogenic acid, caffeic acid, and ferulic acid. The secondary metabolite may include, for example, chlorogenic acid, caffeic acid and ferulic acid.

According to the present invention, it was confirmed that the contents of chlorogenic acid, caffeic acid and ferulic acid, which are excellent in antioxidant, anti-inflammatory, and antibacterial effects, were significantly improved after induction of Roxampire callus. In a specific example according to the present invention, as a result of HPLC analysis of the active ingredient of the in-flight cultured callus extract of Roxampire callus prepared according to the manufacturing method of the present invention, the plant stem cell extract using the callus was overall compared to the adult leaf extract. It was confirmed that the phenolic compound content was increased by about 1.5 times. In particular, it was confirmed that the functional active ingredient, chlorogenic acid, increased by 1.5 times, in the case of caffeic acid by 13.6 times, and in the case of ferulic acid by about 11 times.

With reference to the accompanying drawings, preferred embodiments of the present invention will be described in more detail.

1 is a schematic diagram for explaining a method for producing a loxampire callus for producing a loxampire-derived secondary metabolite according to an embodiment of the present invention.

The present invention comprises a first step of sterilizing and germinating the loxampire seeds and then inducing the plants. In the method according to an embodiment of the present invention, it is important to disinfect the seeds to minimize contamination for aseptic germination and culture when the Roxampire is introduced into the cabin during the first step.

In the case of Roxampire, since the seed shell is double (outer and inner seed coat), the seeds are soaked in water at 4°C for 48 hours, then the outer seed coat is removed using sharp tweezers, and a series of Implementing a disinfection process is important to minimize contamination.

According to an embodiment of the present invention, the disinfection may be performed by immersing the Roxampire seeds in a solution containing 2-5% sodium chlorite (Soduim hypochlorite, NaOCl) and 0.01-0.02% surfactant.

It can be immersed for 15 to 30 minutes for disinfection, but preferably can be disinfected by immersion for 20 minutes under conditions containing 3% sodium chlorate (NaOCl) and 0.01% Tween-20. In some cases, GA3 (Gibberellic acid), a gibberellin-based plant growth regulator, may be used at a concentration of 10 to 100 uM, preferably 10 uM, in order to break dormancy and promote germination of seeds. After seed germination, for growth, it can be transferred to a hormone-free medium to induce rooting and maintain growth.

The present invention includes a second step of inducing callus by culturing the plant tissue or an explant thereof in a medium to which auxin and cytokinin are added. In the method according to one embodiment of the present invention, according to the second step, a tissue of a plant 3 to 5 months old or a section thereof, for example, a tissue from a leaf and a petiole, is cut to a size of about 0.5 cm with a knife, and each section is The sieve was plated on a medium to which growth substances such as auxin and cytokinin were added, for example, MS medium to confirm the rate of induction and proliferation of callus.

As used herein, the term "callus" is an amorphous, undifferentiated cell formed by culturing organs, tissues, etc., as a parenchymal tissue that enlarges and blocks the wound by restoring the dividing ability of the cells of the wound when the plant is wounded. means, and the callus has totipotency to re-differentiate to a complete adult.

According to an embodiment of the present invention, the medium for inducing the callus may be, for example, basic MS or 1/2MS (Half strength MS: 0.5X MS) medium.

The auxin and cytokine are growth regulators, and may be used in combination with auxin and cytokine. The auxin is at least one selected from the group consisting of NAA (1-naphthaleneacetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid), and the cytokinin is 6-BA (6-benzylaminopurine) and kinetin (kinetin). ) may be one or more selected from the group consisting of. Specifically, 2 of auxins containing NAA (1-naphthaleneacetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid) and cytokinins containing 6-BA (6-benzylaminopurine) and kinetin Eggplant or a combination of three growth regulators may be used.

According to an embodiment of the present invention, the following auxin and cytokinin combinations may be used:

A: kinetin, NAA and 2,4-D;

B: 6-BA, NAA and 2,4-D; or

C: 6-BA and NAA.

Callus can be induced by dentin the leaves and petioles of the plant, or explants thereof, in a medium to which one or more combinations of auxins and cytokinins selected from the following group are added. Specifically, callus can be induced by dentin in a medium to which a combination of one or more auxins and cytokinins selected from the group consisting of:

A: Kinetin 0.5 mg/l, NAA 1.5 mg/l and 2,4-D 0.5 mg/l;

B: 6-BA 1 mg/l, NAA 1.5 mg/l and 2,4-D 0.5 mg/l; and

C: 6-BA 1 mg/l and NAA 1.5 mg/l.

According to an embodiment of the present invention, NAA 1.5 mg/l and 2,4-D 0.5 mg/l may be used as the auxin, and kinetin 0.5 mg/l and 6-BA 1 mg/l may be used as the cytokinin. can In leaf and petiole, it was confirmed that callus induction was induced by 80-90% in leaf explants and 40-70% in petiole explants depending on the medium conditions according to the three combinations of hormone composition. In particular, in 0.5X MS medium, NAA 1.5 mg/l, 2,4-D 0.5 mg/l, and 6-BA 1 mg/l were used in a combination of more than 90% in leaf explants and 70% in petiole explants. It showed a high rate of callus induction. In addition, it showed excellent callus induction and growth rate of about 80% in leaf explants and 50% in petiole explants even under the medium combination condition in which 0.5 mg/l of 2,4-D 0.5 mg/l, which is concerned about the harmfulness of the herbicide, was excluded.

According to an embodiment of the present invention, the culture conditions for inducing the callus are 16 hr light conditions/8 hr dark conditions or 24 hr dark conditions, but are not limited thereto.

The present invention includes a third step of selecting and proliferating excellent callus, which can be preserved in a uniform culture with little variation among the callus. In the growth of the callus induced in the second step, an excellent cell line that can be preserved as a uniform culture with little variation was selected and maintained through subcultures 5 to 6 times at 4 week intervals using an optimal medium.

The callus can be propagated by dentition in a medium supplemented with a combination of one or more auxins and cytokinins selected from the following group:

A: kinetin, NAA and 2,4-D;

B: 6-BA, NAA and 2,4-D; or

C: 6-BA and NAA.

According to an embodiment of the present invention, the callus can be propagated by dentition in a medium to which a combination of one or more auxins and cytokinins selected from the group consisting of:

A: Kinetin 0.5 mg/l, NAA 1.5 mg/l and 2,4-D 0.5 mg/l;

B: 6-BA 1 mg/l, NAA 1.5 mg/l and 2,4-D 0.5 mg/l; and

C: 6-BA 1 mg/l and NAA 1.5 mg/l.

According to an embodiment of the present invention, NAA 1.5 mg/l, 2,4-D 0.5 mg/l, and 6-BA 1 mg/l in 0.5X MS medium may be used as an optimal medium for callus proliferation, It is not limited thereto. In some cases, the selected good stock was cultured in a medium containing 1.5 mg/l of NAA and 1 mg/l of 6-BA in 0.5X MS medium before suspension culture in the fourth step, and in culture conditions without 2,4-D. It can be further cultured.

According to an embodiment of the present invention, a fourth step of culturing the proliferated callus is included. Secondary metabolism through suspension culture of the callus grown in the third step in 0.5X MS medium containing 1.5 mg/l of NAA and 1 mg/l of 6-BA for culturing in a liquid medium excluding agar, which is a solid medium. It is possible to increase the content of the phenolic compound as a substance. Culture conditions include, but are not limited to, 16 hr light conditions/8 hr dark conditions or 24 hr dark conditions. In order to investigate the effect of light conditions on the accumulation of active ingredients, the content of secondary metabolites was increased by about 10% overall in the 16 hr light / 8 hr dark conditions with light when culturing callus in 16 hr light / 8 hr dark conditions or 24 hr dark conditions. confirmed to be increased.

Roxampire callus prepared according to the above-mentioned manufacturing method, extract or culture thereof may be used in a cosmetic composition.

Based on this, the present invention relates to a cosmetic composition comprising, as an active ingredient, a loxampire callus, an extract or a culture thereof, prepared by the above preparation method.

The extract is a material separated by dissolving callus in a solvent, and may refer to a product concentrated through distillation or evaporation. The culture medium may refer to a solution containing all useful substances produced or differentiated by culture conditions after culturing the callus.

According to an embodiment of the present invention, the cosmetic composition may be used in the form of a solution, gel, solid, suspension, foam, aerosol or patch.

The cosmetic composition may include other ingredients that are conventionally formulated according to the purpose of the cosmetic. For example, the solution contains preservatives, moisturizers, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, bactericides, antioxidants, stabilizers, glycerin, pH adjusters, alcohols, pigments, fragrances, cooling agents, etc. may be included, but is not limited thereto.

In addition, the cosmetic composition may be supplied to the cosmetic user in the form of a freeze-dried composition. Therefore, cosmetic users can directly apply purified water, lotion, cosmetic liquid, toner, cream, gel, patch, mask, etc. to the freeze-dried composition to apply to the skin.

Example

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Roxampire Seed Germination and Callus Induction Steps

Rocksampire seeds were purchased from a British seed company (Chiltern Seeds, UK), immersed in distilled water for 48 hours and placed at 4° C., and then the outer seed coat of the seeds was removed using sharp tweezers. The seeds from which the outer shell has been removed are immersed in 70% ethanol for 1 minute to remove contaminants, then immersed in 2% sodium chlorite (Soduim hypochlorite, NaOCl) solution containing 0.01% surfactant Tween-20 for 20 minutes, followed by sterilization It was washed three or more times with distilled water. After removing moisture from the surface of the washed seeds using sterilized filter paper, no hormones are added for germination, and MS (Murashige and Skoog, 1952) solid containing 30 g/l of sucrose and 7 g/l of agar. After sowing in the medium, germination is induced at 25°C ± 2°C under light conditions (4000 lux) for one week. After germination, growth was maintained in light/dark conditions (16hr/8hr). After germination, the leaf or petiole of a plant 3 to 5 months old is cut into 0.5 to 1 cm length using a sterile mass, and cytokinin (6-benzylaminopurine (6-BA) or Kinetin) and auxin-based (1-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D)) hormone combinations were treated with three combinations (A to C combinations) as shown in Table 1. The induction and growth rate of callus for each treatment group were observed and specified in Table 1, and the overall schematic diagram for induction of roxampere in-flight and induction of callus in tissues is shown in FIG. 1 . The induced callus was selected and propagated by subculture every 4 weeks.

[Table 1]

^a Callus induction rate was defined as: callus induction rate (%) = (number of explants produced callus/total number of explants cultured) Ⅷ 100%. ^b The callus growth rate was recorded per day basis of callus induction and graded by comparison in four categories as very good (++++), good (+++), poor (++) and very poor (+).

Example 2. Steps for establishing suspension culture from Roxampire callus

To establish a suspension culture system from Roxampire callus, Example 1, a liquid medium of combination C (0.5X MS, NAA 1.5 mg/l, 6-BA 1.0 mg/l, Sucrose 30 g/l) shown in Table 1 Eroxampire callus 5% v/v, that is, 15 g of callus was inoculated into 300 ml of a liquid medium. This culture condition was subcultured 2-3 times for 2 weeks in a 1L Erlenmeyer flask with 2,4-D-free medium, which is a growth hormone that is a problem in commercial application. The culture was performed in suspension at 100 rpm at 25 °C ± 2 °C, white light (4000 lux) for 16 hours, or dark conditions for 24 hours.

Example 3. Preparation of Loxampire Extract

The Roxampire callus obtained by culturing in Example 2 was filtered through a stainless steel filter to separate the culture medium, and after sufficient moisture was removed, it was dried in a dryer at 60° C. for 48 hours. 5 g of dried loxampire plant cell powder was added to 100 ml of purified water, followed by hot water extraction at 120° C. for 1 hour while applying pressure. After extraction, after centrifugation at 4000 rpm for 10 minutes, the solid content was removed, and the supernatant was filtered with a 0.2 μm syringe filter to obtain a finally purified 5% hot water extract.

Example 4. Measurement of Flavonoids and Phenol Contents as Secondary Products

Example 4-1: Determination of total flavonoid content

The total flavonoid content was measured using the Sanaka method (2005) based on the aluminum chloride colorimetric method. After adding 1.25 ml of distilled water to 0.25 ml of each sample of each concentration of the hot-water extracted Roxampire leaf and callus extract and standard material for quantification, _{0.075 ml of 5% sodium nitrate (NaNO 2} ) is added, and mix well. After the reaction at room temperature for 5 minutes, 0.5 ml NaOH (1N) is added, 0.275 ml of distilled water is added to adjust the final volume to 2.5 ml, and then reacted in the dark for 30 minutes. After the reaction, 200 ul of each sample was placed in a 96-well plate in 3 repetitions, and absorbance at 510 nm was measured. Total flavonoid content was quantified using a standard calibration curve prepared in a concentration range of 0 to 500 ug/ml of Catechin (Sigma, C0567) used as a standard material.

Example 4-2: Determination of total phenol content

The total phenol content was measured using Foline-Ciocalteu reagent (Sigma, F9252) to measure the total phenol content of loxampire leaves and callus extracts. Foline-Ciocalteu reagent is a method that is widely used in popular use as a method of reducing the phenolic substances contained in the sample extract to give molybdenum a blue color. After adding 0.9 ml of distilled water to 0.1 ml of each sample of the hot-water extracted Roxampire leaf and callus extract and standard material for quantification, 0.1 ml of 2N Foline-Ciocalteu reagent was added so that the final concentration was 0.2 N. , mix well. After the reaction at room temperature for 5 minutes _{, 1 ml of 7% sodium carbonate (Na 2} CO ₃ ) was added, mixed well for 2 minutes, and then 0.4 ml of distilled water was added to adjust the final volume to 2.5 ml. React in cancer state for 30 minutes. After the reaction, 200 ul of each sample was placed in a 96-well plate, and the absorbance was measured at 750 nm in 3 repetitions. The total phenol content was quantified using a standard calibration curve prepared in the range of 0~500 ug/ml concentration of gallic acid (Sigma, G7384) used as a standard material. The results of flavonoid and phenol content measurement are shown in FIG. 2 and Table 2, respectively.

[Table 2]

Example 5. HPLC analysis

Roxampire callus and leaf extracts obtained in Examples 2 and 3 were diluted with methanol and used as HPLC analysis samples. For HPLC, Thermo Scientific UltiMate3000 series was used, and ultraviolet-visible light (UV-VIS) absorption detector was used. For the column, INNO C18 column (4.6 x 25mm, 0.5um) from Youngjin Biochrom (Korea) was used, and the mobile phase was _{gradient with two solvents, solvent A (0.3% Trifluroaceticacid, TFA in dH 2} O) and solvent B (Acetonitrile). was reacted for 60 minutes. The flow rate was 0.8 ml/min.

As a result of HPLC analysis of the roxampire plant (dried leaf tissue) by the hot water extraction method and the hot water extract according to the culture conditions of the dried callus, chlorogenic acid and its derivatives, a phenolic compound showing antioxidant, anti-aging and anti-inflammatory effects in the callus extract It was confirmed that phosphorus caffeic acid and ferulic acid components were increased (Fig. 3, Table 3, Fig. 4 and Table 4).

[Table 3]

[Table 4]

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby It will be obvious. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.

Claims (7)

a first step of sterilizing and germinating the loxampire seeds and inducing the plants;
The second step of inducing callus by culturing the leaf or petiole of the plant in a medium supplemented with 6-BA 1 mg/l, NAA 1.5 mg/l and 2,4-D 0.5 mg/l ;
A third step of selecting and proliferating excellent callus that can be preserved as a uniform culture with little variation among the callus; and
Chlorogenic acid, caffeic acid, and ferulic acid, including a fourth step of suspending the proliferated callus in a medium containing 1 mg/l of 6-BA and 1.5 mg/l of NAA acid) for producing one or more secondary metabolites selected from the group consisting of loxampire callus.
According to claim 1,
The second step is a method for inducing callus by culturing the leaf or petiole of the plant under light conditions for 16 hours and dark conditions for 8 hours.
delete According to claim 1,
The disinfection is a manufacturing method, characterized in that by immersing the Roxampire seeds in a solution containing 2-5% sodium chlorite (Soduim hypochlorite, NaOCl) and 0.01-0.02% surfactant.
delete A loxampire callus prepared by the method of claim 1 .
A cosmetic composition comprising the Roxampire callus of claim 6, an extract or culture thereof as an active ingredient.

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