KR20220037805A - A method of culturing roots derived from stem cell of Artemisia capillaris Thunb for mass increasing artemisinin - Google Patents

Abstract

The present invention relates to a method for culturing roots derived from an artemisia capillaris thunb stem cell for mass production of artemisinin and an artemisia capillaris thunb stem cell-derived root cultured by the method including the following steps of: a) inducing callus by culturing artemisia capillaris thunb tissue in a medium containing 2,4-D and naphthaleneacetic acid (NAA); b) isolating the stem cell from the callus and culturing the stem cell in a medium containing 2,4-D and NAA; and c) inducing root formation by culturing the stem cell in a medium containing 2,4-D.

Description

A method of culturing roots derived from stem cell of Artemisia capillaris Thunb for mass increasing artemisinin for mass production of artemisinin

The present invention relates to a method for culturing a root culture derived from Artemisinin stem cells for mass production of Artemisinin.

Medicinal crops have been taken for a long time as traditional herbal medicines, have the advantage of less concerns about safety, and are being researched based on modern biotechnology. In particular, medicinal plants have proven efficacy and safety through long clinical experience, reducing failures as biomaterial products and enabling rapid development. Based on these advantages, industrialization is accelerating into medicines and health functional foods made of natural biomaterials using medicinal plants, and 50% of domestic and foreign medicines are single substances derived from natural products. There are 1,253 medicinal plants and 826 edible plants that can be used as medicinal resources in Korea, with a total of 2,104 species growing wild.

Among them, 30 kinds of mugwort native to Korea are used for food and medicinal purposes. Among them, Artemisia capillaris Thunb. has been used since ancient times for fatty liver, chronic hepatitis, gastrointestinal disease, constipation, jaundice, and neuralgia. It is known to have anti-inflammatory, diuretic, and gallstone inhibitory effects.

In particular, Artemisinin is a secondary product of Teroenoids and contains artemisinin, which is well known as a treatment for malaria. It is good for menopausal disorders, and people with a cold body can improve immunity by raising body temperature.

However, since Injin mugwort is expensive in market distribution and difficult to cultivate, it is difficult to mass-cultivate economically because there are few farmers, so there is a crucial limitation in its widespread distribution.

Korean Patent Publication No. 10-2020-0101898

The problem to be solved by the present invention is to provide a method for culturing Artemisinin stem cell-derived root for mass production and a root cultured with Artemisinin stem cell-derived root.

In addition, the present invention is to provide a food composition or a feminine cleanser composition comprising Injin mugwort stem cells, the stem cell-derived root, or a culture solution thereof.

An embodiment of the present invention comprises the steps of: a) culturing Injin mugwort tissue in a medium containing 2,4-D and NAA (naphthaleneacetic acid) to induce callus; b) separating the stem cells from the callus and culturing in a medium containing 2,4-D and NAA (naphthaleneacetic acid); and c) inducing root formation by culturing the stem cells in a medium containing 2,4-D.

The wormwood tissue may be a leaf, a stem, a petiole or a root.

The content of 2,4-D in step a) or b) may be 0.05 to 3.0 mg/L, and the content of NAA may be 0.001 to 0.5 mg/L.

The medium in step a) or b) is a solid batch, and may further include sucrose and gellite.

The culture in step a) or step b) may be darkly cultured at pH 5.5-6.5 and 20-30°C.

The content of 2,4-D in step c) may be 0.01 to 0.1 mg/L.

The medium in step c) may be a liquid medium.

The root may have an artemisinin content of 2 to 5 times that of the Injin mugwort tissue.

Another embodiment of the present invention is Artemisinin (artemisinin) stem cell-derived roots for mass production.

Another embodiment of the present invention is a food composition comprising Injin mugwort stem cells, the stem cell-derived root, or a culture solution thereof.

Another embodiment of the present invention is a feminine cleanser composition comprising Injin mugwort stem cells, the stem cell-derived root, or a culture solution thereof.

Injin mugwort stem cell-derived root culture method of the present invention can be used to culture Artemisinin stem cell-derived roots for mass production. It can be mass-produced and industrialized.

In addition, it is possible to provide a food composition or a feminine cleanser composition comprising Injin mugwort stem cells, the stem cell-derived root, or a culture solution thereof.

1 is a photograph of Injin mugwort callus derived from Injin mugwort according to an embodiment of the present invention.
2 is a photograph of roots derived from Injin mugwort stem cells according to an embodiment of the present invention.
3 is a schematic diagram of the biosynthesis of artemisinin according to the present invention.
4 is a photograph of Injin mugwort callus induced by different culture conditions according to the present invention.
5 is a photograph of roots induced by different culture conditions according to the present invention.
6 is a photograph showing the hydrogen peroxide reaction of stem cells isolated from Injin mugwort callus induced according to the present invention.
7 is a graph comparing the amount of artemisinin biosynthesis of Injin mugwort tissue, stem cells isolated from Injin mugwort callus, and stem cells-derived roots.

Hereinafter, specific embodiments of the present invention will be described in detail so that those of ordinary skill in the art can easily carry out the present invention. However, the present invention may be embodied in several different forms and is not limited to the embodiments described herein.

An embodiment of the present invention comprises the steps of: a) culturing Injin mugwort tissue in a medium containing 2,4-D and NAA (naphthaleneacetic acid) to induce callus; b) separating the stem cells from the callus and culturing in a medium containing 2,4-D and NAA (naphthaleneacetic acid); and c) inducing root formation by culturing the stem cells in a medium containing 2,4-D.

1 is a photograph of Injin mugwort callus derived from Injin mugwort according to an embodiment of the present invention, FIG. 1a is a callus derived from Injin mugwort stem, FIG. , Figure 1d is a callus derived from Injin mugwort root.

2 is a photograph of a root induced from Injin mugwort stem cells according to an embodiment of the present invention, FIG. 2a is a root derived from leaf-derived stem cells, FIG. 2b is a root derived from petiole-derived stem cells, and FIG. 2c is a root derived from stem-derived stem cells.

3 is a schematic diagram of the biosynthesis of artemisinin according to the present invention.

In the callus according to FIG. 1 , when explants are placed on a medium during tissue culture of a plant and cultured under appropriate culture conditions, a cell mass forming a certain system is formed, and this cell mass is called a callus. The callus contains stem cells, and thus stem cells can be separated from the callus.

The Injin mugwort stem cell-derived root according to FIG. 2 may be derived from the Injin mugwort stem cell, and the root contains a high content of artemisinin, and a greater amount of artemisinin than the Injin mugwort stem cell or Artemisinin. contains this.

Artemisinin according to FIG. 3 is a secondary product of teroenoids and is well known as a therapeutic agent for malaria. In addition, the artemisinin is a main component of the excellent antibacterial, antioxidant and anti-inflammatory effects of wormwood, and is good for female poor circulation, menstrual irregularities, and menopausal disorders. etc. can be used.

In the present invention, the Injin mugwort tissue may be a leaf, a stem, a petiole, or a root, preferably a leaf or a stem. The petiole refers to the top part that attaches the leaf body to the stem or branch.

The size of the leaf, stem, petiole or root may be 1-3 mm.

In addition, in the present invention, before applying the Injin mugwort tissue to the medium, a sterilization process of sterilizing the Injin mugwort tissue may be performed.

The sterilization can be performed in the following order, and if the time is prolonged depending on the sterilization treatment time, the tissue is destroyed. It is important to perform sterilization.

First, sterilize for 10 to 60 seconds with 50 to 90% ethanol. Next, the sterilized tissue was washed 1 to 5 times with sterile distilled water, and then immersed in 0.5 to 2% sodium hypochlorite aqueous solution for 5 to 10 minutes. Wash 1-5 times with sterile distilled water to prevent the disinfectant remaining on the epidermis from penetrating into the endothelial cells. Then, it was immersed again in 0.1 to 1% sodium hypochlorite for 3 to 10 minutes, and then washed 3 to 5 times with sterile water.

Step a) is a step of inducing Injin mugwort tissue into a callus, and step b) is a step of isolating stem cells from the callus and subculturing them.

The 2,4-D (2-4-dichlorophenoxy acetic acid) is a type of auxin that is a growth regulator, and the content of 2,4-D in step a) or b) is 0.05 to 3.0 mg/L and may preferably be 2.0 to 3.0 mg/L. When the content of 2,4-D is less than 0.05 mg/L, strong and long roots are formed, and induction into callus is not easy. Separation is not easy.

The NAA (naphthaleneacetic acid) is naphthalene acetic acid and is a kind of auxin, a growth regulator. It is almost the same as indole acetic acid, but because of its high stability, it is used to promote rooting or induce unit deletion.

The content of the NAA in step a) or b) may be 0.001 to 0.5 mg/L, preferably 0.1 to 0.5 mg/L. If the content of NAA is less than 0.001 mg/L, the higher the 2,4-D concentration, the more simple callus is generated, so it may not be easy to separate stem cells. can be induced.

In the present invention, when 2,4-D and NAA are added together in step a) or step b), a synergistic effect occurs, and the effect of culturing stem cells contained in Injinworm callus can be maximized compared to when added alone.

In step a) or b), the medium is a solid batch, preferably MS medium, and the MS medium may have a composition as shown in Table 1 below. In addition, the medium in step a) or step b) may further include sucrose (Sucrose) and gellite (Gerlite).

Ingredient name Content (mg/L) CoCl2.6H2O 0.025 CuSO4.5H2O 0.025 FeNaEDTA 36,70 H3B03 6.20 KI 0.83 MnSO4.H2O 16.90 Na2MoO4.2H2O 0.25 ZnSO4.7H2O 8.60 CaCl2 332.02 KH2PO4 170.00 KNO3 1900.00 MgSO4 180.54 NH4NO3 1650.00 Glycine 2.00 Myo-Inositol 100.00 Nicotinic acid 0.50 Pyridoxin HCl 0.50 Thiamine HCl 0.10

In the present invention, the culture of step a) or step b) may be darkly cultured at pH 5.5-6.5 and 20-30°C.

In addition, in step b), the stem cells may be separated from the callus and subcultured, and in step b), the temperature is controlled to 25° C. or less, preferably 18 to 22° C., and oxygenated to prevent browning of the stem cells. A dentition method to block can be performed.

Step c) is a step of inducing roots from stem cells, and the content of 2,4-D in step c) may be 0.01 to 0.1 mg/L, preferably 0.05 to 0.1 mg/L. . If the content of 2,4-D in step c) is less than 0.01 mg/L or exceeds 0.1 mg/L, induction into roots may not be easy.

In step c), the medium may be a liquid medium, preferably a liquid medium containing 25 to 50% by weight of MS salts. If the MS salt is less than 25% by weight or exceeds 50% by weight, induction from stem cells to roots may not be easy.

In the present invention, the culture of step c) may be darkly cultured at pH 5.5-6.5 and 20-30°C.

In the root cultured according to the present invention, the content of artemisinin may be 2 to 5 times that of Injin mugwort tissue.

As described above, using the culture method according to the present invention, it is possible to culture the roots derived from Artemisinin stem cells for mass production of Artemisinin.

Another embodiment of the present invention is an artemisinin stem cell-derived root for mass production, and the root may have an artemisinin content of 2 to 5 times that of an artemisinin tissue.

Another embodiment of the present invention is a food composition comprising Injin mugwort stem cells, the stem cell-derived root, or a culture solution thereof.

In addition, "culture solution" refers to a cell culture solution remaining after culturing the cells, excluding the cells.

In the present invention, "callus" refers to a cell or a cell mass that has not been differentiated through a dedifferentiation process, and may include stem cells.

The food composition of the present invention includes all types of functional food (functional food), nutritional supplements (nutritional supplement), health food (health food) and food additives (food additives). Food compositions of this type can be prepared in various forms according to conventional methods known in the art.

For example, as a health food, the composition itself may be prepared in the form of tea, juice, and drink to be consumed, or granulated, encapsulated, and powdered for ingestion. In addition, functional foods include beverages (including alcoholic beverages), fruits and their processed foods (eg canned fruit, canned fruit, jam, marmalade, etc.), fish, meat, and their processed foods (eg ham, sausage corn beef, etc.); Breads and noodles (eg udon noodles, soba noodles, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, syrup, dairy products (eg butter, cheese, etc.), edible vegetable oils and fats, margarine, vegetable protein, retort foods, It can be prepared by adding the extract to frozen food and various seasonings (eg, soybean paste, soy sauce, sauce, etc.). In addition, in order to use the composition of the present invention in the form of a food additive, it may be prepared and used in the form of a powder or a concentrate.

The preferred content of the Injin mugwort stem cells of the present invention, the stem cell-derived roots, or a culture thereof may be 0.001 to 50%, preferably 0.1 to 50%, based on the total weight of the food composition.

Another embodiment of the present invention is a feminine cleanser composition comprising Injin mugwort stem cells, the stem cell-derived root, or a culture solution thereof.

The preferred content of the Injin mugwort stem cells of the present invention, the stem cell-derived roots, or a culture thereof may be 0.001 to 10%, preferably 0.1 to 10%, based on the total weight of the feminine cleanser composition.

In addition, the feminine cleanser composition may be composed of a composition generally known in the art.

Hereinafter, specific examples according to the present invention will be described.

Example 1: Injin mugwort stem cell-derived root culture

Injin mugwort used in the present invention was sold from the Institute of Medicinal Resources, Gyeongnam Institute of Agricultural Technology, and was grown in pots in the greenhouse of the Ministry of Biological Resources of the National Academy of Agricultural Sciences. About 8 weeks after cutting, the leaves, stems and roots of plants grown to a length of 30 cm were used.

material sterilization

In order to preserve the endothelial tissue of Injin Artemisia and perfect sterilization of the epidermal tissue, it was immersed in 70% ethanol for 20 seconds, washed 3 times with sterile distilled water, and immersed in 1% sodium hypochlorite aqueous solution for 5 to 10 minutes. . Wash 1-5 times with sterile distilled water to prevent the disinfectant remaining on the epidermis from penetrating into the endothelial cells. Then, it was immersed again in 0.5% sodium hypochlorite for 3 to 10 minutes, and then washed 3 to 5 times with sterile water.

Callus induction culture

Prepare the leaves of Injin mugwort 2~3mm, the petiole prepares the part connecting the leaf and the hypocotyl to 2mm, prepare the stem to be 2mm, and the roots are grown as a plant by culturing the growth point in MS basic medium. was prepared by culturing for 12 weeks and cutting it into 3 mm.

The leaves, petioles, stems and roots were dented in a solid culture medium, and callus induction culture was performed by darkly culturing at 25±1° C., and observing callus formation from about 7 days after culturing the plants for each part. started

For the solid culture medium, 30 g of sucrose and 4 g of gelite were added to the basic MS medium of Table 1, and 2,4-D, NAA, and BAP (benzopyrene), a type of cytokine, were added as shown in Table 2 below. , the induced callus is shown in FIG. 4 .

division 2,4-D (mg/L) NAA (mg/L) BAP (mg/L) Hormone-free medium - - - culture medium 1 0.05 - - culture medium 2 0.1 - - culture medium 3 0.2 - - culture medium 4 0.5 - - culture medium 5 1.0 - - culture medium 6 2.0 - - culture medium 7 3.0 - - culture medium 8 0.1 0.01 - culture medium 9 0.1 0.05 - culture medium 10 0.1 0.1 - culture medium 11 2.0 0.1 - culture medium 12 2.0 0.2 - culture medium 13 2.0 0.5 - culture medium 14 2.0 - 0.05 culture medium 15 2.0 - 0.1 Culture medium 16 2.0 - 1.0

4 is a photograph of Injin mugwort callus induced by different culture conditions according to the present invention, FIG. 4a is a photograph of a stem-derived callus, FIG. 4b is a photograph of a leaf and petiole-derived callus, and FIG. am.

Referring to FIG. 4 , in the solid MS medium without the addition of hormones, differentiation into a complete plant or roots were induced. As a result of treatment with 2,4-D at various concentrations (0.05 to 3 mg/L) in callus induction (culture medium 1 to 7), the callus induction amount at low concentrations of 0.05 to 0.2 mg/L (culture medium 1 to 3) On the other hand, strong and thick roots were formed, and as the concentration increased from 0.5 mg/L or more (culture medium 4 and 5), the amount of callus induction increased while the root formation decreased, showing a tendency to become weak and thin.

In addition, at 2.0~3.0 mg/L (culture medium 6 and 7), root formation was hardly observed, and a white, sticky, but falling, band-forming form, characteristic of stem cells, was observed on the callus.

From this, it was possible to obtain callus containing stem cells in the 2~3mg/L single treatment group with high 2,4-D concentration in each part of the leaf, petiole, stem, and root when Injin mugwort was cultured for each part.

On the other hand, as a result of mixing NAA, 2,4-D was fixed at 0.1 mg/L and NAA was mixed with 0.01 to 0.1 mg/L (culture medium 8 to 10), the higher the NAA concentration, the stronger and thicker roots was formed On the other hand, in the group treated with 2,4-D at 2.0 mg/L and NAA mixed with 0.1 to 0.5 mg/L (culture medium 11 to 13), there was a slight difference by site, but 2,4-D alone treatment group (culture medium 11 to 13) A callus covered with stem cells was obtained more than the media 1 to 7).

In the stem-derived treatment group, callus containing mainly stem cells was formed, whereas in the leaf-derived treatment group, the higher the NAA concentration, the weaker and thinner roots were observed. The root-derived treatment group had a light yellow color and had less callus formation than the stem and leaf.

From this, it can be seen that a synergistic effect appears or inhibits depending on the type of plant, plant part, and the type and concentration of hormone, and affects the induction of callus including stem cells.

On the other hand, in the group treated with 2,4-D and BAP, a representative hormone of the cytokinin system (culture medium 14 to 16), when cultured stem and leaf-derived, 1 mg/L mixed treatment with high BAP concentration (culture medium 16) No reaction appeared. However, in the 0.05~0.1mg/L mixed treatment group (culture medium 14 and 15) with a low BAP concentration, callus was induced in a very small amount. On the other hand, no reaction was observed regardless of the BAP concentration during root-derived culture.

Stem cell isolation and subculture

In order to isolate stem cells from the callus, stem cells (white stem cells that form a cotton-like band that are crushed when rubbed by hand) contained in the callus induced from leaves, stems and petioles were separately isolated. . After that, subculture was carried out, and in order to prevent browning during subculture, the cut side was turned over without contact with oxygen when toothed at 20°C and subcultured.

Stem Cell-derived Root Culture 1

Stem cells were isolated from callus derived from petiole, mouth and stem in the culture media 6 and 7. Thereafter, 2,4-D in a liquid medium (1/2 MS medium and 1/4 MS medium) containing 50% by weight and 25% by weight of MS salts was treated with 0.05 mg/L and 0.1 mg/L, respectively, After dispensing 500ml into a 1L flask, stem cells isolated from the callus were dented and cultured. The medium conditions are shown in Table 3 below, and the culture was 120rpm, 25±1℃. The cancer culture was cultured in suspension for 12 weeks. The induced roots are shown in FIG. 5 .

division 2,4D (mg/L) content in solid culture footstalk leaf stem MS salt content
(weight%) 2,4-D (mg/L) culture medium 17 3.0 O - - 25 0.05 culture medium 18 2.0 - O - 25 0.05 culture medium 19 3.0 - - O 25 0.05 culture medium 20 3.0 O - - 50 0.05 culture medium 21 3.0 - O - 50 0.05 culture medium 22 3.0 - - O 50 0.05 culture medium 23 3.0 O - - 25 0.1 culture medium 24 3.0 - O - 25 0.1 culture medium 25 3.0 - - O 25 0.1 culture medium 26 3.0 O - - 50 0.1 culture medium 27 3.0 - O - 50 0.1 culture medium 28 3.0 - - O 50 0.1

Referring to Figure 5, it was seen that the liquid culture grew faster than in the solid culture, which is that the stem cells separated from the callus are broken into pieces in the liquid culture than in the solid culture, and the surface area is much larger, so that the absorption of nutrients from the medium, This is because the effect of hormone concentration and oxygen supply occur more smoothly.

In the case of stems, stem cells isolated from the callus were growing even in the treatment groups in which 2,4-D was added at 0.05 mg/L and 0.1 mg/L, respectively, and in the 0.05 mg/L treatment group with a low 2,4-D concentration (culture In medium 19), sometimes 1 or 2 roots were formed, and in 1/2 MS medium with a higher MS content than 1/4 medium, roots were formed when the 2,4-D concentration was low (culture medium 22). That is, it was found that the stem-derived liquid culture is effective for the formation and growth of stem cells isolated from the callus rather than the roots.

For leaves, roots were formed regardless of MS concentration and 2,4-D concentration. However, in the case of 1/4 MS medium, 2,4-D 0.1mg/L (culture medium 24), roots were formed to the extent that they were tangled, but at a lower concentration of 0.05mg/L (culture medium 18), roots was formed briefly. Also, when the 1/2 MS, 2,4-D concentration was 0.05 mg/L lower than 0.1 mg/L (culture medium 27), the root formation rate was high enough to become entangled with each other. That is, to induce root formation, it is advantageous to use 1/4 MS containing 0.1 mg/L of 2,4-D or 1/2 MS medium containing 0.05 mg/L of 2,4-D in leaf-derived culture. can be known

In the case of petiole origin, only stem cells isolated from callus were maintained and grown in 1/4 MS, 1/2 MS, and 2,4-D 0.1 mg/l (culture medium 20 and 26), but the 2,4-D concentration was low. At 0.05 mg/L (culture medium 17 and 20), stem cells isolated from the callus were formed, and 3 or 4 long and thin roots were also formed.

Stem cell-derived root culture 2

Stem cells were isolated from callus derived from petiole, mouth and stem in the culture medium 11 to 13. Thereafter, 2,4-D in a liquid medium (1/2 MS medium and 1/4 MS medium) containing 50% by weight and 25% by weight of MS salts was treated with 0.05 mg/L and 0.1 mg/L, respectively, After dispensing 500ml into a 1L flask, stem cells isolated from the callus were dented and cultured. The medium conditions are shown in Tables 4 to 6 below, and the culture was performed at 120rpm, 25±1℃. The cancer culture was cultured in suspension for 12 weeks.

division solid culture medium footstalk leaf stem MS salt content
(weight%) 2,4-D (mg/L) culture medium 29 11 O - - 25 0.05 culture medium 30 11 - O - 25 0.05 culture medium 31 11 - - O 25 0.05 culture medium 32 11 O - - 50 0.05 culture medium 33 11 - O - 50 0.05 culture medium 34 11 - - O 50 0.05 culture medium 35 11 O - - 25 0.1 culture medium 36 11 - O - 25 0.1 culture medium 37 11 - - O 25 0.1 culture medium 38 11 O - - 50 0.1 culture medium 39 11 - O - 50 0.1 culture medium 40 11 - - O 50 0.1

division solid culture medium footstalk leaf stem MS salt content
(weight%) 2,4-D (mg/L) culture medium 41 12 O - - 25 0.05 culture medium 42 12 - O - 25 0.05 culture medium 43 12 - - O 25 0.05 culture medium 44 12 O - - 50 0.05 culture medium 45 12 - O - 50 0.05 culture medium 46 12 - - O 50 0.05 culture medium 47 12 O - - 25 0.1 culture medium 48 12 - O - 25 0.1 culture medium 49 12 - - O 25 0.1 culture medium 50 12 O - - 50 0.1 culture medium 51 12 - O - 50 0.1 culture medium 52 12 - - O 50 0.1

division solid culture medium footstalk leaf stem MS salt content
(weight%) 2,4-D (mg/L) culture medium 53 13 O - - 25 0.05 culture medium 54 13 - O - 25 0.05 culture medium 55 13 - - O 25 0.05 culture medium 56 13 O - - 50 0.05 culture medium 57 13 - O - 50 0.05 Culture medium 58 13 - - O 50 0.05 culture medium 59 13 O - - 25 0.1 culture medium 60 13 - O - 25 0.1 culture medium 61 13 - - O 25 0.1 culture medium 62 13 O - - 50 0.1 culture medium 63 13 - O - 50 0.1 culture medium 64 13 - - O 50 0.1

In the case of solid culture, the 2,4-D concentration was fixed at 2.0 mg/L and NAA 0.1 to 0.5 mg/L mixed treatment (culture medium 11 to 13) caused the roots to become entangled in stem cells isolated from petiole or leaf-derived callus. So many roots were formed (not shown).

Example 2: Hydrogen peroxide response to stem cells isolated from Artemisia callus

In order to confirm that the stem cells isolated from the callus including the Injin mugwort stem cells cultured in Example 1 are stem cells, the stem cells isolated from the Injin mugwort callus cultured in culture medium 11 were treated with hydrogen peroxide, which is shown in FIG. .

Hydrogen peroxide is decomposed into water and oxygen by peroxidase and catalase, which are known to exist in large amounts in cambium-derived young stem cells. Referring to FIG. 6 , as a result of treatment with hydrogen peroxide water on stem cells isolated from Injin mugwort callus separated and cultured in the present invention, water and a large amount of oxygen were generated and the result was covered with air bubbles. It can be seen that the stem cells are cambium-derived Injin mugwort stem cells.

Example 3: Comparison of Artemisinin Content in Injin Artemisia Tissue, Stem Cells Isolated from Injin Artemisia Callus and Stem Cell-derived Roots

In order to analyze the content of artemisinin, liquid chromatography-mass spectrometry (LC-MS/MS) was used.

Specifically, after drying the Injin mugwort tissue, the stem cells and stem cell-derived roots isolated from the Injin mugwort callus cultured in Example 1, each 50 mg dry sample was extracted three times with 5, 25, and 25 ml methanol, and then the extract was added with water 25 ㎖ was added and then phase partitioned twice with 10 mL of hexane to collect the methanol/water layer, concentrated, and re-dissolved in 500 μM methanol to be used as an analysis sample, and to analyze the content of artemisinin in the extract , liquid chromatography-mass spectrometry (LC-MS/MS) was used. The conditions of LC-MS/MS used for the analysis of the artemisinin content of the present invention are shown in Table 7 below.

Artemisinin content of stem cells and stem cell-derived roots isolated from Injin mugwort tissue, Injin mugwort callus is shown in FIG. 7 .

7A is a diagram showing the content of artemisinin in the Injin mugwort tissue, FIG. 7B is a diagram showing the content of artemisinin in stem cells isolated from Artemisia callus, and FIG. It is a diagram showing the content of myshinin (artemisinin).

7a to 7c, the content of artemisinin in the stem cell-derived root is 0.57±0.01 mg/L, which is about 4 times the artemisinin content of 0.14±0.01 mg/L in the Injin mugwort tissue. It can be confirmed, and it can be confirmed that the content of artemisinin in the stem cell-derived root is significantly increased.

In addition, considering the characteristics of stem cells isolated from callus, accumulation of such a high concentration of functional substances can be expected even when continuous mass subculture is promoted.

On the other hand, it can be seen that the biosynthesis amount of artemisinin in stem cells isolated from Artemisia callus is 0.23 ± 0.15 mg/L, which is about 1.8 times that of Injin mugwort tissue.

As described above, the content of artemisinin can be increased by using the root culture method for mass production of artemisinin of the present invention. It can be industrialized by mass production of artemisinin.

In addition, it is possible to provide a food composition or a feminine cleanser composition comprising Artemisinin-containing stem cells, stem cell-derived roots, or a culture solution thereof.

The preferred embodiment of the present invention has been described in detail above. The description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains will understand that other specific forms can be easily modified without changing the technical spirit or essential features of the present invention.

Therefore, the scope of the present invention is indicated by the claims to be described later rather than the detailed description, and all changes or modifications derived from the meaning, scope, and equivalent concept of the claims are interpreted as being included in the scope of the present invention. should be

Claims (11)

a) inducing callus by culturing the Injin mugwort tissue in a medium containing 2,4-D and NAA (naphthaleneacetic acid);
b) separating the stem cells from the callus and culturing in a medium containing 2,4-D and NAA (naphthaleneacetic acid); and
c) inducing root formation by culturing the stem cells in a medium containing 2,4-D;
Artemisinin (artemisinin) Injin mugwort stem cell-derived root culture method for mass production comprising a. According to claim 1,
The Artemisinin stem cell-derived root culture method for mass production of Artemisinin, characterized in that the Injin mugwort tissue is a leaf, a stem, a petiole or a root. According to claim 1,
Injin mugwort for mass production of artemisinin, characterized in that the content of 2,4-D in step a) or b) is 0.05 to 3.0 mg/L, and the content of NAA is 0.001 to 0.5 mg/L Stem cell-derived root culture method. According to claim 1,
In step a) or b), the medium is a solid medium, and artemisinin is a method for mass production of Artemisinin stem cell-derived root culture, characterized in that it further comprises a solid medium. According to claim 1,
The culture in step a) or step b) is an artemisinin stem cell-derived root culture method for mass production, characterized in that cancer culture at pH 5.5-6.5 and 20-30°C. According to claim 1,
In the step c), the content of 2,4-D is 0.01 ~ 0.1 mg/L Artemisinin (artemisinin) mass production Injin mugwort stem cell-derived root culture method, characterized in that. According to claim 1,
Artemisinin (artemisinin) mass production of Artemisinin stem cell-derived root culture method for mass production, characterized in that the medium in step c) is a liquid medium. According to claim 1,
The root culture method derived from Artemisinin stem cells for mass production of Artemisinin, characterized in that the content of Artemisinin is 2 to 5 times that of Injin Artemisia tissue. Artemisinin (artemisinin) stem cell-derived roots for mass production cultured by the culture method of any one of claims 1 to 8. A food composition comprising Injin mugwort stem cells, the stem cell-derived root, or a culture solution thereof. A feminine cleanser composition comprising Injin mugwort stem cells, the stem cell-derived root, or a culture solution thereof.

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