KR101765599B1 - Anti-inflammatory Composition Comprising Plant Stem Cell Derived from Cambium of Family Ginkgoaceae - Google Patents

Abstract

The present invention relates to an anti-inflammatory composition containing cambium-derived stem cell derived from cambium, an extract thereof, a lysate thereof, and a culture thereof. The composition according to the present invention has been found to have excellent anti-inflammatory effects against a mouse model in which ulcerative colitis has been induced using DDS, and thus it is useful as a composition for the prevention and treatment of inflammatory diseases.

Description

[0001] The present invention relates to an anti-inflammatory composition comprising cambium-family stem cells derived from cambium as an active ingredient,

The present invention relates to an anti-inflammatory composition containing cambium-derived stem cell derived from cambium, an extract thereof, a lysate thereof, and a culture thereof.

Inflammation is a local reaction to the wound site to initiate entry of pathogens or removal of injured tissue. Despite the positive role of inflammation, it has become one of the most common onset mechanisms for human disease. The production of nitric oxide (NO) by activated monocytes and macrophages initiates a potent inflammatory response and is the most important part of the initial immune response to bacterial pathogens (Bogdan C., Nat . Immunol ., 2 : 907, 2001).

Inflammatory reactions are caused by various inflammatory mediators and immune cells in the local blood vessels and body fluids when they are infected with tissue (cell) damage or external infectious agents (bacteria, fungi, viruses, various kinds of allergens) It exhibits a series of complex physiological responses such as substance secretion, fluid infiltration, cell migration, and tissue destruction, and external symptoms such as erythema, edema, fever, and pain. In normal cases, the inflammatory reaction removes the external infectious agent and regenerates the damaged tissue to regenerate the organism's function. However, if the antigen is not removed or the internal substance causes the inflammatory reaction to occur excessively or continuously, Some lead to diseases such as cancer. Thus, there has also been a need for the development of natural anti-inflammatory agents that can prevent excessive and persistent inflammatory reactions without side effects.

Therefore, the inventors of the present invention have found that the anti-inflammatory effect of the cambium-derived stem cells of the ginkgo and Ginkgo biloba-derived stem cells and the culture medium thereof has excellent anti-inflammatory effects as a result of efforts to minimize the side effects of the existing anti-inflammatory drugs and to develop a natural- And the present invention was completed.

It is an object of the present invention to provide a natural-material-derived composition showing anti-inflammatory activity.

In order to achieve the above object, the present invention provides a stem cell derived from cambium, which is derived from a cambium layer and has not undergone differentiation, and at least one of cambium-derived stem cells, an extract thereof, Lt; / RTI >

The present invention also provides a functional food for preventing or ameliorating inflammation comprising at least one of cambium-derived stem cells, an extract thereof, a lysate thereof, and a culture thereof.

The present invention has an effect of providing an anti-inflammatory composition containing as an active ingredient any one or more of cambium-derived stem cells, extracts thereof, lysates thereof and cultures thereof. The composition according to the present invention has been found to have excellent anti-inflammatory effects against a mouse model in which ulcerative colitis has been induced using DDS, and thus it is useful as a composition for the prevention and treatment of inflammatory diseases.

Fig. 1 is a photograph of a cambium layer observed from a cross section of a material plant (ginkgo).
FIG. 2 is a photograph showing induction and isolation of stem cells according to the present invention, wherein 2A shows extremely different forms of stem cells (arrow) and phloem cells (star table) This is a picture 3 weeks later (scale bar: 1 mm).
FIG. 3 is a photograph (scale bar: 0.25 mm) of a stem cell (A) according to the present invention and a solid culture of bark cut bursa-derived callus (B).
FIG. 4 is a micrograph (× 100, scale bar: 50 μm) observing the degree of cell aggregation of callus (B) derived from stem cells (A) and bark of the ginkgo in this culturing process.
FIG. 5 is a photomicrograph of stem cells (B) and gingko somatic cells (B) according to the present invention, and the scale bar at the lower left is 15 μm.
FIG. 6 is a micrograph of a stem cell (A) according to the present invention and a callus (B) derived from a ginkgo bark tissue obtained after staining a vacuole using Neutral red, and the scale bar at the lower right side is 25 μm.
FIG. 7 is a photograph showing the mitochondria of callus (B) derived from stem cell (A) and ginkgo bark tissue according to the present invention, wherein the scale bar at the lower left is 15 μm.
FIG. 8 is a graph showing cell growth curves in a bioreactor of a stem cell according to the present invention. FIG.
FIG. 9 is a graph showing the proliferation rate of callus derived from stem cells and ginkgo biloba according to the present invention.
10 is a photograph showing a scale-up process in a 3 L air floating biological reactor (A), a 20 L air floating biological reactor (B), and a 250 L air floating biological reactor (C).
11 is a graph showing cell survival rate after cryopreservation of callus derived from stem cells and ginkgo biloba according to the present invention.
FIG. 12 shows the results obtained by performing column chromatography on the ethanol extracts of stem cells according to the present invention and obtaining the seven fractions having 120 different fractions having similar patterns through TLC analysis.
FIG. 13 is a graph showing cell viability after MTT analysis of stem cell extract fraction according to the present invention in RAW 264.7 macrophage cell line (** p <0.01).
FIG. 14 is a graph showing inhibition of NO activity (* p <0.05, ** p <0.01, *** p <0.001) by measuring the nitrite concentration after treatment with the stem cell extract fraction according to the present invention in RAW 264.7 macrophage cell line .
FIG. 15 shows the amounts of iNOS and COX-2 gene expression by LPS treatment after treatment with the stem cell extract fraction according to the present invention in RAW 264.7 macrophage cell line.
16 is a photograph showing the degree of activation of ERK and p38 MAP kinase by Western blotting by LPS treatment after treatment with a stem cell extract fraction according to the present invention in RAW 264.7 macrophage cell line.
FIG. 17 is a photograph showing the degree of IκB degradation and NF-κB activation by LPS treatment after Western blotting treatment of RAW 264.7 macrophage cell line according to the present invention.
FIG. 18 is a graph showing the inhibitory effect of IL-6 (A) and TNF-α on LPS-treated stem cell extract fraction treated with RAW 264.7 macrophage cell line according to the present invention (* p <0.05, ** p &Lt; 0.01).
19 is a graph showing GSH concentration (A), SOD activity (B), catalase activity (C), and GPx activity (D) in normal control, vitamin C and stem cell extract treatment according to the present invention between mice * p < 0.05, ** p < 0.01, *** p < 0.001).
20 is a graph showing ALT activity (A), ALP activity (B), AST activity (C) and LDH activity (D) in normal control, vitamin C and stem cell extract treatment according to the present invention in mouse blood * p < 0.05).
Figure 21 shows the effects of cambium on cambium-derived stem cells (CMCs) on intestinal inflammation and body weight in the DDS inflammation model.
FIG. 22 shows the effect of cambium on cambium-derived stem cells (CMCs) on the colonic IgA concentration in the DDS inflammation model (* p <0.05, ** p <0.01).
FIG. 23 is a graph showing the effect of cambium stem cell (CMCs) extract on cytokine concentration in MLN lymphocyte culture (* p <0.05, ** p <0.01, *** p <0.001).
24 is a graph showing the effect of cambium stem cells (CMCs) extracted from cambium on blood IgG and IgA concentrations (* p < 0.05).
FIG. 25 is a graph showing the effect of cambium stem cell (CMCs) extract on cytokine expression in splenocytes (* p <0.05, ** p <0.01, *** p <0.001).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

The definitions of the main terms used in the description of the present invention and the like are as follows.

In the present application, the vascular "cambium" is a mesenchymal tissue located within the vascular tissue of a plant and located in the stem and root. The activity of the cambium causes the growth of plants, resulting in the presence of large plants with an annual ring of more than 11,000 years. The development of the anatomical structure of the anatomical structure is the same as that of the same organism (Fig. 10, 1993) In the invention, the cambium layer is interpreted to include a procambium layer. It is a matter of course that the same effect is obtained by using the cambium layer and the procambium layer structure in the present invention as the cambium and cambium layer being the same first divisional tissue.

As used herein, the term "lysate" means a cell lysate obtained by disrupting a cell by a chemical method or a physical method using detergent or the like. The "extract" of a cell line is a substance obtained by dissolving a cell in a solvent and using distillation or evaporation &Lt; / RTI &gt; The term "culture solution" of a cell line refers to a cell culture solution remaining after cells are cultured. In addition, the term "culture product" in the present application is a concept including a culture solution and / or a cultured cell line, wherein the cultured cell line includes all cell lines that are differentiated by culture conditions or have improved productivity and / .

The term "stem cell " as used herein refers to a congenitally undifferentiated cell that is genetically more stable and does not undergo a process of dedifferentiation.

The term " innately undifferentiated "as used herein refers to maintaining the pre-differentiation state, rather than being undifferentiated through a dedifferentiation process.

"Callus" herein refers to a cell or cells in non-differentiated state through a dedifferentiation process, lumps (Proc Natl Acad Sci USA, 99 (25):.... 15843, 2002) refers to.

In one aspect, the present invention relates to an anti-inflammatory composition containing at least one of a cambium stem cell derived from cambium, an extract thereof, a lysate thereof, and a culture thereof.

The cambium-derived stem cells derived from the ginkgoaceae according to the present invention are derived from cambium-derived cambium and are undifferentiated, undifferentiated, cells. It may be characterized in that it has at least one of the following characteristics:

(a) contains a large number of single cells or a small-sized cell aggregate as compared to the dedifferentiated callus of Ginkgo biloba during suspension culture;

(b) exhibits morphological features with multiple vacuoles;

(c) has an increased activity of mitochondria compared to dedifferentiated callus of Ginkgo biloba;

(d) Growth rate is faster and longer than that of demineralized callus from ginkgo; And

(e) Has low sensitivity to shear stress in bioreactors as compared to demineralized callus from Ginkgo biloba.

The term "having a plurality of vacuoles " means that the cells have a plurality of vacuoles more than twice as much as those derived from a callus derived from a dedifferentiated callus. In addition, the stem cells according to the present invention have small vacuoles in size in comparison with cells derived from callus of Ginkgo biloba. In addition, "having a number of advanced forms of mitochondria" refers to a multiplicity of more than two-fold greater activity of mitochondria under an optical microscope BX41 (Olympus, Japan) as compared to cells derived from demineralized callus from ginkgo .

In the present invention, the stem cells may be characterized in that they have at least two or more of the characteristics of (a) to (e), and preferably at least 3 or more of the characteristics of (a) to (e) , And more preferably at least four of the characteristics (a) to (e). In addition, in the present invention, the stem cells may be characterized by having all of the characteristics (a) to (e).

On the other hand, the stem cells according to the present invention comprise (a) obtaining a cambium-containing tissue from a ginkgo plant; (b) culturing the obtained cambium-containing tissue in a medium; And (c) separating the cells from the cambium layer to obtain cambium-derived stem cells, wherein (b) comprises culturing the cambium-containing tissue to grow cambium The cambium layer of cambium-derived stem cells may be derived by separating the cambium layer from the cambium layer of step (c). In addition, the step (b) may be performed in a culture medium containing auxin, wherein the auxin is selected from the group consisting of NAA (alpha -naphthalene acetic acid), IAA (indole-3-acetic acid) Picloram may be used, and such auxin may be contained at a concentration of 1 to 5 mg / L. In the step (c), cambium layer is separated from a callus layer grown amorphously from a portion other than the cambium layer to obtain cambium-derived stem cells.

In the present invention, the culture may be prepared by culturing the stem cells in an amount of 3-5% (g / L) of raw sugar or sugar as an elicitor; Or further culturing in a culture medium containing at least one of methyl jasmonate, chitosan, phenylalanine, benzoic acid, ABA, salicylic acid and sodium acetate. . [0042] The above-mentioned &quot; Preferably, the medium comprises 3-5% (g / L) of sugar or sugar; And methyl jasmonate, fungal extracts, bacterial extracts, yeast extracts, chitosan, glucomanan, glucan, phenylalanine, benzoic acid, salicylic acid ABA, SNP, IPP, BHT, CCC, ethephon, hippuic acid, ceric ammonium nitrate, and the like. material selected from the group consisting of AgNO _3, vanadyl sulfate (vanadyl sulfate), p- amino acid benzo (p -aminobenzoic acid), bra Sino steroid (brassinosteroids), sodium alginate (sodium alginate), sodium (sodium acetate) ethyl And a culture medium.

In the present invention, it is also possible to use a culture obtained by subjecting the stem cells to ultraviolet rays, heat, ethylene, an antifungal agent, an antibiotic, a heavy metal salt and a high salt concentration as an elicitor and applying physical and chemical stress thereto.

In the present invention, the extract may be extracted using a solvent selected from the group consisting of distilled water, alcohol, acetone, dimethyl sulfoxide (DMSO), and a mixed solvent thereof.

In an embodiment of the present invention the Ginkgo bank Araliaceae bank hawthorn (genus Ginkgo) (Ginkgo It has confirmed that the anti-inflammatory activity by biloba) separating the cambium-derived stem cells of Ginkgo from, and thus the present invention is a preferably a bank hawthorn (genus Ginkgo) uses the cambium-derived stem cells of a plant, more preferably from Ginkgo Stem cells derived from cambium can be used.

In the examples of the present invention, it was confirmed that the stem cell extract according to the present invention and its fractions have antioxidative and anti-inflammatory activities in vivo as well as in vitro. In particular, it was confirmed that the stem cells according to the present invention exhibit a very excellent anti-inflammatory effect in a mouse model of ulcerative colitis caused by DDS.

Accordingly, even though there is no specific example in which the composition containing the stem cell lysate or the culture medium exhibits anti-inflammatory activity, it has been confirmed that the stem cells and the extract have anti-inflammatory activity as described above, It will be apparent to those skilled in the art that the stem cell according to the present invention or a composition containing the lysate may exhibit anti-inflammatory activity to prevent or treat an inflammatory disease.

The anti-inflammatory composition containing the stem cell, the extract thereof, the lysate thereof, and the culture thereof according to the present invention as an active ingredient may be used alone or in combination with one or more pharmaceutically acceptable carrier, excipient or diluent And the stem cells can be contained in the pharmaceutical composition in an appropriate pharmacologically effective amount depending on the disease and its severity, the patient's age, body weight, health condition, sex, administration route and treatment period, etc. have.

The term "pharmaceutically acceptable" as used herein refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction such as a gastrointestinal disorder, dizziness, or the like when administered to a human. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Polyvinyl pyrrolitone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition may further include a filler, an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifier, and an antiseptic agent. The pharmaceutical compositions of the present invention may also be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders.

In another aspect, the present invention relates to a functional food for preventing or ameliorating inflammation, which contains, as an active ingredient, at least one of stem cells, an extract thereof, a lysate thereof, and a culture thereof according to the present invention.

Herein, the term &quot; functional food &quot; means that the function of the food is improved by adding at least one of the stem cells or the extract, the lysate and the culture thereof according to the present invention to the general food.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

In particular, in the following examples, antioxidative and anti-inflammatory effects of cambium stem cells derived from cambium, extracts thereof and fractions thereof were confirmed. However, it is known in the art that similar results can be obtained by using the stem cell lysate or culture medium It is obvious to those who have the knowledge of.

Obtain cambium stem cells from cambium

1-1: Preparation of plant material

Ginkgo and Ginkgo Ginkgo biloba , Buyeo, Korea), as shown in Fig. 1, can be observed through phloroglucinol staining to reveal xylem and phloem fibers, and cambium is present therebetween. In order to obtain the cambium-derived stem cells from such ginkgo, the stem of the ginkgo was firstly harvested and immediately immersed in a solution of 100 mg / L ascorbic acid (DUCHEFA, The Netherlands) Respectively. At this time, in order to obtain the same stem cells using the procambium layer, twigs are collected instead of stem.

Then, 1% benomyl (Dongbu Hannong Chemical, Korea), 1% daconil, Dongbu Hannong Chemical, Korea, 1% streptomycin (DUCHEFA, The Netherlands), 0.1% cefotaxime sodium DUCHEFA, The Netherlands) for 24 hours and then washed with tap water for 30 minutes to remove phenolic compounds and residual chemicals. After sterilization for 1 min in 70% ethanol (DC Chemical, Korea), 15 min in 30% hydrogen peroxide (LG Chemical, Korea), 15 min in 1% CLOROX solution and 5 min in 3% CLOROX solution And washed 3 to 4 times.

1-2: Formation layer from stem Fragmentary  Isolate manufacturing tissue

The bark tissue containing the formation layer of the stem (the twig in the case of using the procambium layer) subjected to the sterilization process of Example 1-1 was lifted and easily peeled off from the xylem. The peeled tissues constitute an explant comprising the cambium, phloem, cortex, and epidermis.

1-3: Derivation of cambium stem cells from cambium

The cambium-containing slices prepared in Example 1-2 were cultured in the stem cell induction medium (medium 1) shown in Table 1.

Cell line induction medium (medium 1) Composition Contents (mg / L) Inorganic salts

KNO ₃ 2500 (NH _{4) 2} SO ₄ 134 MgSO ₄ .7H ₂ O 121.56 MnSO ₄ · 4H ₂ O 10 ZnSO _{4 ·} 7H ₂ O 2 CuSO ₄ · 5H ₂ O 0.025 CaCl ₂ .2H ₂ O 113.23 KI 0.75 CoCl ₂ .6H ₂ O 0.025 NaH ₂ PO ₄ · H ₂ O 130.44 H ₃ BO ₃ 3 Na ₂ MoO ₄ .2H ₂ O 0.25 FeNaEDTA 36.7 Vitamin
Myo-inositol 200 Thiamine-HCl 20 Nicotinic acid 2 Pyridoxine-HCl 2 L-ascorbic acid 50 Citric acid 75 Amino acid


L-aspartic acid 133 L-arginine 175 Glycine 75 Proline 115 Hormone picloram One Sucrose 10,000 Activated charcoal 100 Gelrite 2,000

Auxin such as NAA, IAA, and picloram may be added to the medium at a concentration of 1 to 5 mg / L, preferably 1 mg / L. Cultivation was carried out in a dark room controlled at 25 ± 1 ° C.

On the 4th to 7th days of the initial culturing, cell division was visually observed from the cambium layer, and after 15 days from the cultivation, amorphous callus induced by dedifferentiation was induced from the calcia, cortex and epidermis. After 30 days of cultivation, the cultured cambium layer began to separate from the layer containing the calcaneus, that is, the amorphous callus layer (Fig. 2A: stem cell (arrow), pholem cell (star)), When complete separation was achieved, the cells were separately cultured in different petridishes. After the separation, the white, loose portion having a good growth rate was subcultured every 14 days with the same new medium as the induction medium. Fig. 2B is a photograph of the cambium-derived stem cells after they are separated and cultured for 3 weeks.

On the other hand, for comparison, bark sections of Ginkgo biloba were disinfected and cultured in the medium of Table 1. As shown in Fig. 3B, the bark fragment was transformed into callus In the case of The callus derived from the bark fragment was amorphous due to the difference in the rate of cleavage between several cells such as calcified tissues, and the growth rate was unstable and easily browsed. The callus induced by browning and agglomerated bark fragment was ultimately dulled by the phenolic compounds secreted by it and eventually necrotized. Namely, callus derived from bark was difficult to maintain and cultivate after 6 months. On the other hand, as shown in FIG. 3A, stem cells derived from the cambium-derived cambium were stably maintained in the growth rate, growth pattern, and degree of coagulation of the cells in a long-term culture, enabling mass culture.

1-4: Growth and characterization of cambium stem cells derived from cambium

The cambium-derived stem cells isolated in Example 1-3 were placed in a flask containing the liquid medium of Table 2, and cultured in a shaker at 100 rpm at 25 ± 1 ° C under dark conditions. The subculture period was fixed to 2 weeks, so that the cultured cells were always able to maintain high vigor in the algebraic growth state.

Suspension medium (badge 2) Composition Contents (mg / L) Inorganic salts

Ca (NO _{3) 2} 471.26 NH ₄ NO ₃ 400 MgSO ₄ .7H ₂ O 180.54 MnSO ₄ · 4H ₂ O 22.3 ZnSO _{4 ·} 7H ₂ O 8.6 CuSO ₄ · 5H ₂ O 0.25 CaCl ₂ .2H ₂ O 72.5 K ₂ SO ₄ 990 Na ₂ MoO ₄ .2H ₂ O 0.25 H ₃ BO ₃ 6.2 KH ₂ PO ₄ 170 FeNaEDTA 36.7 Vitamin
Myo-inositol 200 Thiamine-HCl 20 Nicotinic acid 2 Pyridoxine-HCl 2 L-ascorbic acid 50 Citric acid 75 Amino acid


L-aspartic acid 133 L-arginine 175 Glycine 75 Proline 115 Hormone picloram One Sucrose 30,000

As shown in FIG. 4A, the stem cell according to the present invention includes a large number of single cells in suspension culture, and a small number of cell aggregates in suspension culture under an optical microscope BX41 (Olympus, Japan) But the somatic cells (callus derived from the bark tissue) of the control group, Ginkgo biloba, were observed to aggregate as shown in Fig. 4B.

Meanwhile, as shown in FIG. 5A, the stem cells according to the present invention were able to observe a morphological characteristic having a plurality of vacuoles. This feature is a characteristic of the undifferentiated cells existing in the plant due to causes such as pressure and the like, and thus it can be confirmed that the stem cells according to the present invention are undifferentiated. In the case of somatic cells of the common ginkgo (callus derived from bark tissue), such characteristics could not be confirmed, as shown in Fig. 5B. As a result of staining the vacuole with neutral red, the stem cells according to the present invention were able to identify a plurality of red vacuole, as shown in FIG. 6A, and were able to identify somatic cells Tissue-derived callus), as shown in FIG. 6B, it was confirmed that one large central vacuole was present.

Meanwhile, the stem cells according to the present invention were observed through an optical microscope BX41 (Olympus, Japan), and a number of mitochondria highly active on the movement plane could be observed. FIG. 7A shows that stem cells according to the present invention have a plurality of mitochondria, and arrows indicate mitochondria. On the other hand, in the case of somatic cells (callus derived from bark tissue) of common ginkgo, these characteristics could not be confirmed as shown in Fig. 7B.

On the other hand, in order to measure the growth rate per culture period, the growth rate of the cell culture was measured using an air floating bioreactor (Samsung Science, Korea) having a volume content of 3 L. As a result, When we cultured for 1 week, it was able to confirm the growth rate of 2 times and 3 weeks for 2 weeks. At this time, the medium used in the liquid medium of Table 2 was kept at 25 ± 1 ° C under the dark condition. In addition, the callus derived from gingko bark tissue and the cambium stem cell derived from Ginkgo biloba according to the present invention were cultured in a 3L air floating type bioreactor under the same conditions and conditions, and compared with each other.

Growth Rate and Growth Index (GI) Cell lines Growth rate (folds) GI Stem Cell 3.1 2.27 Callus 2.3 1.3

As a result, as shown in Table 3 and FIG. 9, the growth rate of callus derived from gingko bark tissue was 2.3 times, and the growth index (GI) (growth index = (maximum DCW - initial DCW) / initial DCW) Was found to be 1.3. However, it was found that the cambium-derived stem cells according to the present invention had a growth rate of 3.1 times and a growth index (GI) of 2.27, which was higher than that of callus derived from ginkgo biloba. Generally, in the case of a reactor, the cell viability is rapidly reduced due to the generation of a growth ring in the reactor, sensitivity to shear stress due to cohesion of the plant culture and cell wall during culturing, but the stem cell culture Formed a very small area of growth rings in the bioreactor, and the ring on the inner wall was easily solved by moving the medium with a simple stimulus in the incubator. In addition, the cohesion was small and the cell viability was hardly decreased due to its low sensitivity to shear stress due to its large vacuole.

In a scale-up process, Bioreactors Max. dry cell weight (g / L) A 3L air-lift bioreactor 11.3 B 20L air-lift bioreactor 10.1 C 250L air-lift bioreactor 11.7

In addition, as shown in Table 4 and FIG. 10, when the cells were cultured in a 3 L air-floating bioreactor, a 20 L air-floating bioreactor and a 250 L air-floating bioreactor for 10 days, And the amount of dry cell per liter was increased in the case of mass culture of 250 L.

That is, it was confirmed that the cambium-derived stem cells according to the present invention have a low sensitivity to shear stress in a bioreactor for mass culture, so that rapid mass growth is possible in a bioreactor. Thus, it was found that the cambium-derived stem cells according to the present invention had a lower sensitivity to shear stress than the dedifferentiated callus-derived cell line of Ginkgo biloba.

On the other hand, the callus derived from the ginkgo biloba and the stem cells derived from the ginkgo biloba according to the present invention were cryopreserved. Suspension cultures were used for 6 to 8 days of incubation. The cryopreservation medium was a medium containing 0.5 M glycerol (DUCHEFA, The Netherlands), 0.5 M DMSO (DUCHEFA, The Netherlands) and 1 M sucrose (DUCHEFA, The Netherlands) , And transferred to 5 mL cryovial (Duran, USA). The amount of cells inoculated into the cryopreservative is 200 mg / mL. The cryopreserved suspension cells were kept in a freezer for 30 minutes, then stored in a deep freezer for 3 hours, then immersed in liquid nitrogen for freezing.

After that, cultured cells kept in liquid nitrogen for more than 20 minutes were taken out for thawing, placed in a constant temperature water bath at 40 ° C, and thawed for 1 to 2 minutes. For cell regeneration, the cell suspension was used as a sterilized funnel and filter paper. The filtered cells were applied onto a solid growth medium containing filter paper, stabilized at room temperature for 30 minutes, and then transferred again to fresh solid growth medium.

Survival rate measurement results during cryopreservation Cell lines sample 1 sample 2 sample 3 Average survival rate (%) Stem cell
33/212 43/218 45/211 18.87
15.60% 19.70% 21.30% Callus
5/175 4/124 4/158 2.87
2.90% 3.20% 2.50%

As a result, as shown in FIG. 11 and Table 5, it was confirmed that the callus induced from the ginkgo bark tissue had a very low survival rate as compared with the stem cells derived from the ginkgo seedlings of the present invention.

In addition, the characteristic features of plant stem cells include pluripotency in addition to self renewal ability. In order to induce the differentiation of stem cells from gingko cambium-derived stem cells, it was confirmed that the ductal factor was differentiated from cambium-derived stem cells by culturing under MS medium containing growth regulator at 25 ± 1 ° C. .

1-5: Elicitor's  process

Stem cells cultured for 14 days in a medium supplemented with 3-5% (g / L) of raw sugar and 100 μM of methyl jasmonate were added to sterilized water for 14 days as in Example 1-4. The following experiment was conducted.

Production of ethanol extract of cambium stem cells derived from cambium

2-1: Preparation of ethanol extract

(I) 50 g of 80% ethanol was added to 2 g of the stem cells isolated in Example 1 from which the culture medium had been removed, and the mixture was shaken at 15 DEG C for 48 hours.

(Ii) The supernatant produced after shaking was lyophilized to obtain an ethanol extract.

2-2: DMSO  ( dimethyl sulfoxide ) Preparation of extract

(I) 500 g of the stem cells isolated in Example 1, from which the culture solution had been removed, 500 mL of DMSO was added and dissolved at 50 DEG C for 6 hours with stirring.

(Ii) After the dissolution, the supernatant was taken by centrifugation at 3,000 x g for 10 minutes to obtain a DMSO-soluble substance.

(Iii) The DMSO-soluble material obtained above was concentrated using a rotary vacuum concentrator.

(Iv) Concentrated samples were dried using a freeze dryer to obtain DMSO extract.

2-3: Preparation of distilled water extract, methanol extract and acetone extract

From the stem cells prepared in Example 1, the active substances were extracted in the following steps.

(I) 500 g of the cell line isolated in Example 1 from which the culture solution was removed was dissolved in 500 mL of distilled water while stirring at 50 DEG C for 6 hours.

(Ii) After the dissolution, the supernatant was taken by centrifugation at 3,000 x g for 10 minutes to obtain a distilled water-soluble substance.

(Iii) After obtaining the distilled water-soluble substance, 500 mL of methanol was added to the remaining distilled water-insoluble matter, and the mixture was stirred and dissolved at room temperature for 6 hours.

(Iv) After the dissolution, the mixture was centrifuged at 3,000 x g for 10 minutes to obtain a supernatant, thereby obtaining a methanol-soluble substance.

(V) After obtaining the methanol-soluble substance, 500 mL of acetone was added to the remaining methanol insoluble matter, and the mixture was stirred and dissolved at room temperature for 6 hours.

(Vi) After the dissolution, the mixture was centrifuged at 3,000 xg for 10 minutes to obtain an acetone-soluble substance.

(Ⅶ) The distilled water, methanol, and acetone soluble material obtained above were concentrated using a rotary vacuum concentrator.

(Ⅷ) Concentrated samples were dried using a freeze dryer and dissolved in cell culture medium, methanol and acetone to obtain distilled water extract, methanol extract and acetone extract.

Preparation of Extract Fractionated Samples of Stem Cells Derived from Cambium

A fraction sample was prepared from the ethanol extract prepared in Example 2-1 as follows.

To prepare the fractions from the ethanol extracts, column chromatography (Sephadex LH-20 column, 15 x 1260 mm, bead volume: 222 mL) was performed. Gel filtration was performed using Sephadex LH-20 (150 × 1260 mm, 100% methanol) to separate the active material from the prepared 4.2 g of 80% ethanol extract of CMCs. And a flow rate per minute of 0.8 ml (SV = 0.23) to obtain a total of 120 fractions. Each fraction was analyzed by thin layer chromatography (TLC) using 7 fractions (from Fr. I to Fr. VII) (Fig. 12).

Fr.I (# 1): (Fr. 1-7), 2.4612 g

Fr.II (# 2): (Fr. 8-11), 0.199 g

Fr.III (# 3): (Fr.12-15), 0.141 g

Fr. IV (# 4): (Fr. 16-23), 0.2265 g

Fr. V (# 5): (Fr. 24 to 30), 0.1785 g

Fr. VI (# 6): (Fr.31-60), 0.3036 g

Fr. VII (# 7): (Fr.61-120), 0.2329 g

Extract fraction of cambium stem cells derived from cambium in vitro  Identify antioxidant effect

4-1: Hypoxanthine / xanthine oxidase assay Antioxidant activity of fractionated samples

(30 mM EDTA (pH 7.4), 30 mM xanthine (CMC)) and the extracts of the extracts of Cambium meristem cells (CMCs) obtained from Example 2-1 or the Ginkgo biloba prepared in Example 3 , 1.42 mM NBT (nitro blue tetrazolium)) was added to 50 mM PBS, followed by addition of xanthine oxidase (0.5 U / mL) for 15 minutes. The absorbance of the solution was measured at 560 nm, and the extent of the antioxidative effect of the fractionated sample was measured (the degree of inhibition of superoxide radical formation) compared with the control group. As a control, the absorbance of xanthine oxidase treated without antioxidant treatment was determined by measuring the absorbance and the superoxide radical scavenging activity of each fraction was determined as a percentage.

Superoxide radical scavenging activity (unit:% inhibition) of stem cell extract fractions from cambium of cambium density
(쨉 g / mL) #One #2 # 3 #4 # 5 # 6 # 7 extract 10 8.7 18.2 70.1 81.6 80.8 74.4 56.0 50.5 100 12.5 72.6 71.9 100 100 100 100 73.9 1000 95.4 69.2 100 100 100 100 100 100

As a result, as shown in Table 6, when the concentration of 10 μg / mL was used in the fractions # 3, # 4, # 5 and # 6 of the seven fraction samples, the inhibition effect was over 70% And the superoxide radical scavenging activity.

4-2: Deoxyribose assay Of the fractionated sample hydroxyl radical scavenging  activity measurement

100 μM FeCl ₃ and 104 μM EDTA (pH 7.4) were added to the glass tube, followed by reaction for 10 minutes. Then, 1.5 mM hydrogen peroxide, 2.5 mM deoxyribose, and 100 μM vitamin C were added thereto and the ethanol extract of Example 2-1 or Example 3 (CMCs) extracts from the cambium of each ginkgo were added and incubated at 37 ° C for 1 hour. Each reaction sample was mixed well with 1.5% 2-TBA (2-thiobarbituric acid) and 2.8% TCA (trichloroacetic acid), reacted at 80 ° C for 30 minutes and absorbance was measured at 532 nm. Deoxyribose is decomposed by hydroxyl radicals generated by Fenton system (Fe ^{3 +} -ascorbate-EDTA-H ₂ O ₂ ) and malonaldehyde is produced by heating under acidic conditions. The resulting malonaldehyde reacts with thiobarbituric acid to form a pink chromogen, which increases the absorbance. Therefore, in the presence of hydroxyl radical scavenging activity in the sample, decomposition of deoxyribose is inhibited by the addition of the sample and the absorbance decreases. The hydroxyl radical scavenging activity of the sample was expressed as the inhibition rate of hydroxyl radical formation by the change of absorbance of the sample addition group based on the change of absorbance of the group without addition of the sample to 100%.

The hydroxyl radical scavenging activity (unit:% inhibition) of fractionated samples of cambium stem cell extracts density
(쨉 g / mL) #One #2 # 3 #4 # 5 # 6 # 7 extract 10 0 0 0 0 0 0 0 36.7 100 27.8 19.8 63.2 78.5 83.6 74.7 86.7 48.3 1000 63.9 69.3 100 100 100 100 100 100

As a result, as shown in Table 7, when the concentration of 100 μg / mL was used in the fractions # 4, # 5, # 6 and # 7 of the seven fraction samples, it was found that the fraction had more than 70% hydroxyl radical scavenging activity there was.

Forming the origin of ginkgo on the basis of these results stem cells (CMCs) are common in a high assay of extracts from two to seven fraction samples vitro # 4, # 5, and # 6 fraction in the authentic sample showing the antioxidant activity were selected as the target of the antiinflammatory effect study in vitro .

Survival rate of cambium stem cells from cambium-derived stem cells by treatment with extract fraction

The in vitro antiinflammatory effect of the extract layer fractions of cambial stem cell (CMCs) extracts of Example 3 was confirmed using a mouse macrophage cell line RAW 264.7 cell line. To determine the cytotoxicity of each fraction sample, cell viability was investigated using 3- (4,5-dimethylthiazole-2yl-) - 2,5-diphenyl tetrazolium bromide (MTT).

First, each fraction sample and crude extract (CE) of Example 2-1 were first treated at a concentration of 50 μg / mL and then treated with LPS at a concentration of 1 μg / mL for 24 hours. As shown in FIG. 13, the survival rate of the cells treated with MTT assay was about 90% in the CE treatment group and in the LPS treatment group, and about 50% in the each fraction treatment group, as shown in FIG.

This means that the cytotoxicity of each fraction is slightly cytotoxic compared to the cambium stem cell (CMC) extract (CE) of Ginkgo biloba, and it is presumed that some toxicity due to the enrichment effect of the active ingredient, or incidentally, It was judged to be shown. Therefore, it is considered necessary to determine the exact amount of application of the fraction extract or active ingredient in order to harmonize the physiologically active ingredient.

Identification of Anti-Inflammatory Activity of Extracted Fraction of Safflower Stem Cells from Ginkgo

6-1: LPS of RAW  264.7 macrophage cell line  Process nitric  oxide ( NO ) Stem cells from the cambium of cambium CMCs ) Inhibitory effect of extract fraction fractions

LPS-treated macrophage cells have been widely used as useful models for measuring inflammatory responses. In particular, inflammation-related cytokines, MAP kinases, and NF-kB activation are measured as indicators in this model. In particular, NO is the most fundamental measure of the inflammatory response in the macrophage cell line treated with LPS, since it is increased by iNOS, an indicator of various inflammatory responses. Each of the fraction samples of Example 3 and crude extract of Example 2-1 was first treated at a concentration of 50 μg / mL and then treated with LPS at a concentration of 1 μg / mL for 24 hours. The amount of NO was confirmed by measuring the nitrite concentration

As a result, as shown in FIG. 14, the cambium stem cell (CMC) extract (CE) and each fraction were treated at a concentration of 50 μg / mL in a RAW 264.7 macrophage cell line, When LPS was added for 24 hours, it was found that the concentration of NO induced by LPS decreased to a statistically significant level (SigmaPlot, t- test ) by treatment with all extract fractions as well as CMCs CE.

From the results shown in FIG. 13, there is a fear that the anti-inflammatory effect of the CMCs may be toxic to the cells because each fraction sample showed weak cytotoxicity on the macrophage cell line used in the experiment. However, The cytotoxicity of CMCs extracted from cambium stem cells from ginkgo trees was significantly inhibited by inhibition of NO activity (Fig. 14) and MTT assay (Fig. 13) It was not the cause. Therefore, stem cells from cambium-derived stem cells (CMCs) were expected to have an effective anti-inflammatory activity.

6-2: LPS of RAW  264.7 macrophage cell line  The expression of cyclooxygenase-2 ( COX -2) and iNOS  For the gene CMCs  Inhibitory effect of extract fraction

As described above, the expression of NO in most inflammatory reactions is an important indicator, and the expression of NO is known to be caused by the action of iNOS enzyme. Therefore, expression of iNOS gene is an essential element of inflammatory response measurement. COX-2 is also known to be one of the most important indicators of inflammatory response. Therefore, we investigated how the iNOS and COX-2 genes expressed in the RAW 264.7 macrophage cell line treated with LPS were inhibited by the cambium-derived stem cell (CMCs) extract fraction, CMCs) extract fraction.

Then, each fraction sample of Example 3 and crude extract (CE) of Example 2-1 were firstly treated at a concentration of 50 μg / mL in RAW 264.7 cells and treated with 1 μg / mL of LPS to obtain 24 And the amounts of COX-2 and iNOS expressed thereafter were confirmed by RT-PCR.

At this time, RT-PCR was performed as follows.

First, the cultured RAW 264.7 cells were collected in an eppendorf tube, washed with PBS, and total cellular RNA was extracted using a total RNA extraction kit. First, Trizol was added and the cells were lysed at room temperature for 5 minutes. Then, chloroform was added and vortexed at room temperature for 15 seconds. After 2-3 minutes, centrifugation was carried out at 12,000 rpm at 4 ° C for 15 minutes. The supernatant was carefully taken, isopropanol was added, and the reaction was carried out at room temperature for 10 minutes. The RNA was centrifuged at 12,000 rpm for 10 minutes at 4 ° C, Abandoned. RNA was extracted from the RNA pellet by centrifugation (at 12,000 rpm for 5 min at 4 ° C) using 80% ethanol, air-dried, and DEPC H ₂ O. After confirming the total RNA by electrophoresis, first strand cDNA was synthesized by quantifying RNA using a spectrophotometer.

Then, cDNA synthesis was performed by adding 0.5 μg oligo dT primer to total RNA (2 μg), reacting at 72 ° C for 5 minutes, adding a reverse transcriptase mixture (5x RT buffer, 25 mM MgCl ₂ , 2 mM dNTP , RNase Inhibitor, RTase) was added and reaction conditions were set in the order of 5 minutes at 25 ° C, 60 minutes at 42 ° C, and 15 minutes at 70 ° C to synthesize cDNA.

Then, put a specific primer as described below with 10 mM dNTPs, 10x Ex-Taq TM polymerase buffer, 2.5 U Ex-Taq TM DNA polymerase in 1 mg cDNA was amplified cDNA. The primer sequence used for the amplification of the target gene is as follows.

COX-2 gene

Forward primer: 5'-AAG AAG AAA GTT CAT TCC TGA TCC C-3 '(SEQ ID NO: 1)

Reverse primer: 5'-TGA CTG TGG GAG GAT ACA TCT CTC C-3 '(SEQ ID NO: 2)

iNOS gene

Forward primer: 5'-CTG CAG CAC TTG GAT CAG GAA CCT G-3 '(SEQ ID NO: 3)

Reverse primer: 5'-GGG AGT AGC CTG TGT GCA CCT GGA A-3 '(SEQ ID NO: 4)

GAPDH gene

Forward primer: 5'-CCG ATG CCC CCA TGT TTG TG-3 '(SEQ ID NO: 5)

Reverse primer: 5'-GGC CAT GCC AGT GAG CTT CC-3 '(SEQ ID NO: 6)

The cycling condition is first reacted at 95 ° C for 2 minutes. When the temperature falls below the melting temperature (Tm), primer dimer formation or non-specific initiation occurs. The PCR products were incubated at 72 ° C for 10 min. The PCR products were stored at 4 ° C for 30 cycles of three-step program (95 ° C for 1 min, 55 ° C for 1 min, and 72 ° C for 1.5 min).

10 μL of the PCR product was electrophoresed on a 1% agarose gel containing 0.2 mg / mL ethidium bromide. The amplified DNA band was analyzed by AlphaDigiDoc 1200 software and the degree of expression was analyzed using the GAPDH gene The relative amount was obtained.

As a result, as shown in Fig. 15, the degree of expression of iNOS and COX-2 gene was significantly increased in RAW 264.7 cell treated with 1 mg / mL of LPS compared to the negative control. However, the expression of iNOS and COX-2 gene was significantly decreased when 50 μg / mL of 80% ethanol extract of cambium stem cells (CMCs) from Ginkgo biloba was treated. As a result, iNOS was 57.1 %, And COX-2 was 83.4% less than the LPS control group.

In addition, it was found that 69% to 100% of iNOS and COX-2 gene expression was inhibited by treatment with cambium stem cells (CMCs) extract fraction (50 μg / mL). These results indicate that these samples have an anti-inflammatory effect, as shown by the inhibition of NO production in the CMCs extract fraction sample of Fig. 14 of Example 6-1.

6-3: LPS of RAW  264.7 macrophage cell line  Depending on the treatment MAP kinase  activation of stem cells from cambium CMCs ) Inhibitory effect of extract fraction fractions

It is known that LPS is treated in macrophage cells to produce various mediators involved in the inflammatory reaction. These mediators also include inflammatory cytokines, which require signaling stimulation by LPS, and MAP kinase activation is considered to be an important step in this process. Therefore, activation of MAP kinase by LPS treatment was inhibited by CMCs And it was confirmed that it was inhibited by the treatment of the extracted fraction sample.

Each fraction sample of Example 3 and the crude extract of Example 2-1 were first treated at a concentration of 50 μg / mL in RAW 264.7 cells, treated with LPS at a concentration of 1 μg / mL and incubated for 30 minutes. The inhibitory effect of MAP kinase was confirmed by Western blotting.

The RAW 264.7 cells cultured in the above were collected in a micro centrifuge tube, washed with PBS, and then lysed in a solution buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 2 mM EDTA, 1% NP- Cells were lysed with 10 mM VaF, 1 mM Na3VO4, 10 mM sodium pyrophosphate, 1 mM PMSF, 10 mg / mL aprotinin, 10 mg / mL leupeptin and 0.1 mg / mL soybean inhibitor) Respectively. Each protein was quantitated by Bradford method using BSA as a standard sample. Proteins were dissolved in Laemmli sample buffer (0.625 M Tris-HCl, pH 6.8, 5% β-mercaptoethanol, 2% SDS) and 20 μg of lysate was denatured by 10% SDS-PAGE. The gel was immersed in 0.45 mm hydrophobic PVDF difluoride) membrain (Hybond-P). PVDF was blocked with Tris buffered saline (TBS; 20 mM Tris / HCl, 137 mM NaCl, pH 7.6) containing 5% skimmed milk for about 2 hours and then over-reacted with primary antibody (1: 500) And then washed three times with TBS. Thereafter, the reaction was carried out at room temperature for 1 hour with a HRP-conjugated anti-rabbit secondary antibody solution (dilution ratio 1: 1,000). After washing the same again as described above, X- . As a control for the expression of each protein, the same method as above was carried out using the primary antibody against β-actin.

As a result, as shown in FIG. 16, activation of ERK and p38 MAP kinase was significantly increased by treatment with 1 μg / mL LPS in RAW 264.7 macrophage cell line. It was also found that 27.4% of ERK and 37.3% of p38 MAP kinase inhibited MAP kinase activation in the treatment of cambium stem cells (CMCs) extracted from 50 μg / mL of Ginkgo biloba. It was shown that MAPK activation induced by LPS was also inhibited by treatment of cambium stem cells derived from cambium stem cells (CMCs). Particularly, fraction # 4 had 50% inhibitory activity against LPS control Could know. As with the NO, iNOS, and COX-2 results, CMCs It is a result of consistently proving the anti-inflammatory effect of the extracted fraction samples.

6-4: LPS of RAW  264.7 macrophage cell line  Depending on the treatment NF - kB activation Derived stem cells from gingko CMCs ) Inhibitory effect of extract fraction fractions

Treatment of LPS with macrophage cells produces a variety of mediators that are involved in the inflammatory response. In order to produce inflammatory cytokines such as these mediators, LPS stimulation signals must be transmitted and MAP kinase activation is an important intermediate step in this process. Activation of NF-kB by activation of MAP kinase is known to be the final determinant of gene expression. Therefore, it was confirmed that the degradation of IkB by LPS treatment and the subsequent activation of NF-kB were inhibited by the treatment of cambium-derived stem cells (CMCs) extracted fraction.

Each fraction sample of Example 3 and the crude extract of Example 2-1 were first treated at a concentration of 50 μg / mL in RAW 264.7 cells, treated with LPS at a concentration of 1 μg / mL and incubated for 1 hour. The inhibition of p-IkB and NF-kB activity was confirmed by Western blotting.

The experiment of p-IkB was the same as that of Example 6-3. NF-kB was performed using NE-PER Nuclear & Cytoplasmic Extraction Reagents kit (Thermo science, Rockford, USA).

The RAW 264.7 cells cultured in the above were collected in a microcentrifuge tube, washed with PBS (12,000 rpm for 5 min), and then vortexed for 15 sec with ice-cold Cytoplasmic extraction reagent I (CER I) The ice condition was maintained for 10 minutes. The ice-cold Cytoplasmic extraction reagent II (CER II) solution was vortexed for 5 seconds and kept in ice for 1 minute. The tubes were vortexed for 5 seconds, then quickly transferred to a 4 ° C micro-centrifuge and centrifuged at 12,000 rpm for 5 minutes. The cytoplasmic extract was transferred to a pre-cooled tube and stored at -80 ° C.

The remaining pellet fraction is vortexed vigorously for 15 seconds with ice-cold Nuclear Extraction Reagent (NER) and vortexed vigorously vigorously vigorously at intervals of 10 minutes (4 total). The completely dissolved solution was centrifuged at 12,000 rpm for 15 minutes and the nuclear extract was transferred to a pre-cooled tube and the following experiment was carried out. Each protein was quantitated by Bradford method using BSA as a standard sample. Proteins were dissolved in Laemmli sample buffer (0.625 M Tris-HCl, pH 6.8, 5% β-mercaptoethanol, 2% SDS) to remove 20 μg of lysate by 10% SDS-PAGE. The gels were resuspended in 0.45 mM hydrohhobic PVDF difluoride) membrain (Hybond-P). PVDF was blocked with Tris buffered saline (TBS; 20 mM Tris / HCl, 137 mM NaCl, pH 7.6) containing 5% skim milk for about 2 hours and then over-reacted with primary antibody (1: 500) And then washed three times with TBS. Thereafter, the reaction was carried out at room temperature for 1 hour with a HRP-conjugated anti-rabbit secondary antibody solution (dilution ratio 1: 1,000). After washing the same again as described above, X- . As a control for the expression of each protein, the same method as above was carried out using the primary antibody against β-actin.

As a result, as shown in FIG. 17, the treatment with 1 μg / mL of LPS significantly increased the degradation by IkB phosphorylation and NF-kB activation in the RAW 264.7 macrophage cell line. In addition, NF-kB activation was inhibited by 10.7% when treated with 50 μg / mL gambia stem cells derived from cambium-derived stem cells (CMCs). Interestingly, it was found that treatment of the cambium stem cells (CMCs) derived from the cambium extract significantly inhibited the activation of NF-kB induced by LPS. The degree of inhibition was about 70% there was. This is consistent with the results of inhibition of NO, iNOS, COX-2, and MAP kinase activation.

6-5: LPS of RAW  264.7 macrophage cell line  Inflammation due to treatment cytokine  Expression of cambium stem cells from cambium CMCs ) Inhibitory effect of extract fraction

Treatment of LPS with macrophage cells produces a variety of mediators that are involved in the inflammatory response. The most important factor is the production of inflammatory cytokines. We confirmed that the production of IL-6 and TNF-a, the most important inflammatory cytokines, by LPS stimulation was inhibited by treatment of cambium stem cells (CMCs) extracted fraction.

In order to investigate the inhibitory effect of CMCs extracted from cambium stem cells on inflammatory cytokine production, each fraction sample of Example 3 and a crude extract of Example 2-1 were dissolved in RAW 264.7 cell in a concentration of 50 μg / mL, followed by treatment with LPS at a concentration of 1 μg / mL for 48 h. Cell culture was then measured by ELISA.

First, each cytokine capture antibody was added to a 96-well ELISA plate and reacted overnight at 4 ° C. Plate was washed 5 times with 1x PBS (PBS-T) containing 0.05% Tween-20 and then blocked with 1x assay diluent solution containing 2% BSA for 2 hours at room temperature. The plate was washed three times with 1x PBS-T, and the cell culture medium or each cytokine standard protein was added thereto, followed by reaction at room temperature for 2 hours. The cells were washed 5 times with 1x PBS-T and incubated for 2 hours at room temperature. The plate was washed 5 times with 1x PBS-T, and the avidin-HRP solution was added and reacted at room temperature for 30 minutes. After washing with 1 × PBS-T solution for 7 times, TMB substrate solution was added and the reaction was allowed to proceed for 15 minutes at room temperature. The reaction was terminated by adding a stop solution (1 MH ₃ PO ₄ ). Plate reactivity was measured by absorbance at 430 nm in a microplate reader.

As a result, RAW 264.7 cells were treated with 50 μg / mL of the ethanol extract of cambium stem cells derived from cambium (CMCs) and a sample, respectively, and then 1 μg / mL of LPS was added thereto. As expected, the levels of IL-6 and TNF-a in the culture medium were higher than those in the LPS-treated group, -a expression was significantly decreased.

Using these results in a deferred cost a fraction extract prepared from the cambium-derived stem cells (CMCs) an extract of Ginkgo LPS-stimulated RAW 264.7 macrophage cell In vitro antiinflammatory effect test showed that it has very good antiinflammatory effect.

Stem cell extracts from cambium-derived cambium in vitro  Antioxidant effect measurement

In addition to measuring the antioxidant activity of the cambium stem cell extract fraction in Example 4, the antioxidant activity of cambium stem cells derived from cambium was evaluated by Xanthin / xanthin oxidase assay and deoxyribose assay Respectively.

7-1: Xanthin / xanthin oxidase  Analysis of inhibitory activity effect

Xanthin / xanthin oxidase system have been widely used to generate the O ^.- and HO in the study of oxidative stress caused by free radicals. Xanthin / Xanthin oxidase is widely distributed from bacteria to humans and is one of the enzymes known as molybdenum iron-sulfur flavin hydroxylase. Xanthin oxidase is an enzyme involved in the in vivo purine metabolism. It forms uric acid in xanthin or hypoxanthin and causes gout. As the uric acid is excessively produced in the blood, it is deposited in joints, joint tissues and kidneys to cause inflammation, This causes pain and kidney disease. Xanthin / Xanthin oxidase oxidizes hypoxanthine to xanthine, which is then oxidized to uric acid, which produces O ₂ ^- and H ₂ O ₂ . X / XO system in vitro In experiments ^O. ₂ ^- and H ₂ O ₂ can be used to measure the activity of damage below the cellular level by reactive oxygen species or its defense mechanism (especially enzymatic defense).

To determine Xanthin oxidase inhibitory activity, the reaction mixture (30 mM EDTA (pH 7.4), 30 mM xanthine, 1.42 mM NBT (nitro blue tetrazolium)) and 50 mM PBS were added to the ethanol extract of Example 2-1 xanthine oxidase (0.5 U / mL) was added and reacted for 15 minutes. The absorbance of the solution was measured at 560 nm and the degree of inhibition of the antioxidative effect of the sample was measured in comparison with the control. As a control, the absorbance of xanthine oxidase treated without antioxidant treatment was determined by measuring absorbance, and the superoxide radical scavenging of each fraction was converted into a percentage.

Superoxide radical scavenging activity (unit:% inhibition) of cambium stem cell (CMCs) extract and vitamin C Concentration (mg / mL) CMCs extract Vitamin C One
10
50
100
500 27.78
30.95
53.97
73.8
85.72 35.4
42.86
55.24
63.18
56.58

7-2: Deoxyribose assay

First, non-site-specific hydroxyl radical formation assays were performed. That is, 100 μM FeCl ₃ and 104 μM EDTA (pH 7.4) were placed in a glass tube and reacted for 10 minutes. Then, 1.5 mM hydrogen peroxide, 2.5 mM Deoxyribose and 100 μM vitamin C were added thereto. The cambium-derived stem cell (CMCs) ethanol extract was added and reacted at 37 ° C for 1 hour. Each reaction sample was mixed well with 1.5% 2-TBA (2-thiobarbituric acid) and 2.8% TCA (trichloroacetic acid), reacted at 80 ° C for 30 minutes and absorbance was measured at 532 nm.

The site-specific hydroxyl radical formation assay was then performed as follows. All the procedures were the same as the non-site-specific hydroxyl radical formation assays described above except that instead of vitamin C, the same amount of phosphate buffer was added.

OH for 2-deoxyribose, a sugar ^. The reaction of the compound produces many products. Deoxyribose is decomposed by hydroxyl radicals generated by Fenton system (Fe ^{3 +} -ascorbate-EDTA-H ₂ O ₂ ) and malonaldehyde (MDA) is produced by heating at the acid state (low pH). The resulting malonaldehyde reacts with thiobarbituric acid (TBA) to form a pink chromogen (2TBA-MDA), which shows the maximum absorbance at 532 nm. Therefore, in the presence of hydroxyl radical scavenging activity in the sample, decomposition of deoxyribose is inhibited by the addition of the sample and the absorbance decreases. In this model system, the presence of ascorbic acid, H ₂ O ₂ , Fe (III) -EDTA, and 2-deoxyribose coexist ^. The rate constant of the scavengers can be measured and compared, but the condition is imposed that there should be no reaction between these scavengers and these constituents. The hydroxyl radical scavenging activity of the samples was expressed as the percent inhibition of hydroxyl radical formation by changing the absorbance of the sample-added group based on the absorbance change of 100% of the sample-free addition group (Table 9).

As the concentration of cambium stem cell (CMCs) extract increased, the site-specific, ie, scavenger effect of divalent metal ion was increased in proportion to the concentration of non-site specific (hydroxyl radical).

The deoxyribose assay (% inhibition) of cambium stem cells (CMCs) CMCs concentration
(mg / mL) Site-specific
(% inhibition) Non site-specific
(% inhibition) Control
One
10
50 0.00
4.00 ± 0.05
54.28 ± 0.08
93.14 + 0.12 0.00
13.00 + 0.06
19.19 ± 0.11
44.12 + - 0.10

Stem cells derived from cambium CMCs ) Of the extract in vivo  Antioxidative effect

Based on the results of cambial stem cell-derived stem cells (CMCs) extract of Example 7, in In order to test vivo antioxidant effect, vitamin C, a representative antioxidant, was tested as follows.

The liver is an important organs responsible for nutrients and metabolism and detoxification. It induces liver damage in experimental animals and humans due to various stresses and toxins in the body. As a result, stem cells (CMCs) The effect of scavenging of free radicals and the inhibition of peroxide formation were studied. We tried to demonstrate the antioxidant effect of the cambium stem cell extracts from the ginkgo using experimental and experimental approaches such as enzymatic activity of liver function and antioxidant enzyme activity using mouse serum and liver tissue.

8-1: Experimental animal breeding and Experimental group

The cambium-derived stem cells (CMCs) in the extract of Ginkgo biloba For the vivo antioxidant effect, 18 female ICR mice of 4 weeks old were purchased from Orient (Daejeon, Korea). Experimental animals were maintained in feeder room under constant conditions (temperature: 21 ± 2 ℃, humidity: 50-60%, darkness: 12 hour period (07: 00 ~ 19: 00) After adaptation, it was used in the experiment. Six ICR mice were divided into three groups: normal control group, cambium stem cells (CMCs), and Vitamin C group. In the normal control group, 0.5 mL of distilled water was orally administered for 3 weeks. In the experimental group, the ethanol extract of cambium-derived stem cells (CMCs) or vitamin C from the cambium of Example 2-1 was administered at a concentration of 200 mg / kg once every three days Dissolved in distilled water, and administered orally in 0.5 mL increments for 3 weeks.

8-2: Sampling

Before the disruption of the cervical vertebrae, 12 hours before the start of the experiment, blood was collected from the orbit of the mouse to obtain serum, and the blood was allowed to stand for 30 minutes at room temperature. The supernatant (serum) obtained by centrifuging the coagulated blood at 3,000 rpm for 15 minutes at 4 ° C was collected and stored at -80 ° C in a cryogenic freezer. After removing the liver from the cervical dislocation, the mice were washed with cold physiological saline, and the liver tissues were sieved and washed three times to remove the blood. 0.5 g of tissue was taken and each tissue was placed in a homogenize tube. Potassium phosphate buffer (pH 7.3) was added and homogenized with a homogenizer for 5 minutes. This homogenate was centrifuged (20,000 x g , 20 minutes) at 4 ° C and the supernatant was taken and used as a sample.

8-3: Stem cells derived from cambium of cambium using liver tissue CMCs ) Antioxidant Activity Analysis of Extracts

The cambium-derived stem cells (CMCs) in the extract of Ginkgo biloba The antioxidant enzyme GSH concentration, SOD activity, catalase activity, and GPx activity were measured in the liver tissue collected in Example 8-2 as a basic study for the vivo antioxidant effect analysis.

Lipid induced lipid peroxidation interacts with GSH and thiols - containing proteins, and this interaction leads to oxidative stress - induced reduction of GSH and inactivation of thiols - containing proteins. Most of the major enzymes in the cell are oxidized to the thiols needed for activation, which destroys the activity of the enzyme. GSH, which is known as a reactive oxygen scavenger in vivo, is converted to GSSH by removing hydrogen peroxide by GPx and GSSH is reduced to GSH by consuming NADPH by gluthathion reductase (GR) And plays an important role in protecting cells from active oxygen. To investigate the effect of cambium stem cells (CMCs) extracted from ginkgo biloba on the amount of GSH, we measured changes in GSH levels in liver tissues using a gluthathion concentration assay kit from Biovision.

As a result, as shown in Table 10 and FIG. 19A, it was confirmed that GSH content of cambium stem cells derived from cambium-derived stem cells (CMCs) and Vitamin C was significantly higher than that of normal control group. This is the result of proving that cambium stem cell (CMCs) extracts from cambium have the effect of eliminating active oxygen in vivo.

On the other hand, intracellular SOD (superoxide dismutase) is the first line of defense against the cell damage of oxygen free radicals. It converts superoxide anion generated in the oxidation process into hydrogen peroxide and rapidly decomposes the peroxide to protect cells from free radicals. It is the first enzyme that removes active oxygen, and acts to prevent or treat diseases related to active oxygen. To investigate the effect of cambium stem cells (CMCs) extracts on the activity of SOD, the activity of SOD enzyme in liver was measured by SOD activity in hepatic tissue using Superoxide dismutase assay kit of Cayman Chemical Company Respectively.

As a result, as shown in Table 10 and FIG. 19B, it was confirmed that the cambium-derived stem cell (CMCs) group and Vitamin C group were significantly more active than the normal control group. As a result of the above GSH contents, it is a result of proving that cambium stem cells derived from cambium-derived stem cells (CMCs) have the effect of eliminating active oxygen in vivo.

In addition, Catalase is a hemoprotein with four trivalent iron ions (Fe +++) per molecule and is present in an oxidized state during enzyme activation. When SOD removes active oxygen, hydrogen peroxide is generated. This hydrogen peroxide is the final product of the aerobic glycolysis and oxidation process. This enzyme catalyzes the hydrolysis of hydrogen peroxide to remove reactive oxygen species first. To investigate the effects of cambium stem cells (CMCs) extracts on catalase activity, catalase activity in liver tissues was measured by using Invitrogen's Amplex Red catalase assay kit Respectively.

As a result, as shown in Table 10 and FIG. 19C, there was no change in administration of vitamin C group and cambium stem cells derived from cambium-derived stem cells (CMCs) compared to the normal control group. According to antioxidants, when SOD removes active oxygen, there are substances that act on both catalase and GPx enzyme, GPx enzyme only, and catalase enzyme only. Vitamin C and CMCs pass through GPx pathway and remove hydrogen peroxide It is seen as a substance to give.

On the other hand, the excess hydrogen peroxide produced by SOD by reactive oxygen species is detoxified by the catalase and GPx (gluthathion peroxidase) activity in the body, whereas the catalase activity is the primary protective enzyme of hydrogen peroxide, while the GPx removes hydrogen peroxide This gluthathion peroxidase is an enzyme that clears away some sort of active oxygen debris. That is, it is a substance that plays a major role in removing hydrogen peroxide that has the potential to become active oxygen at any time. GPx also serves to repair and repair damaged cells in their original state. To investigate the effect of cambium stem cells (CMCs) extracts on GPx activity, the activity of GPx enzymes in liver tissues was measured using the gluthathion peroxidase assay kit from Cayman Chemical Company. Respectively.

As a result, as shown in Table 10 and FIG. 19D, it was confirmed that stem cells of cambium-derived stem cells (CMCs) and vitamin C group were significantly higher than normal control group. This is the result of proving that the cambium stem cell (CMCs) extract from the cambium has the effect of removing hydrogen peroxide in vivo.

Antioxidant enzyme activity in mouse liver Group GSH (mg / mL) SOD (U / mL) Catalase (arbitary units) GPx (nM / min) Normal control 0.5 ± 0.04 0.59 + 0.06 921.1 ± 59.3 21.2 ± 0.52 Vitamin C 1.4 ± 0.19 0.99 ± 0.06 1091.2 ± 14.1 27.5 ± 0.29 CMCs 1.2 ± 0.24 0.88 + 0.04 1495.5 ± 23.2 27.1 ± 0.52

8-4: Mouse  Ginkgo biloba using serum CMCs  Biochemical enzyme activity analysis of extract

Serum from the mouse of Example 8-2 was assigned to Green Cross to measure the liver hematological indexes ALT, AST, ALP and LDH. The results are shown in Table 11 and FIG.

The activity of cytotoxic enzyme (* p <0.05) in extracts of cambium stem cells (CMCs) Group ALT (U / L) ALP (U / L) AST (U / L) LDH (U / L) Normal control 43.0 ± 2.4 71.2 ± 4.5 57.8 ± 6.0 432.8 ± 102.2 Vitamin C 38.5 ± 5.0 73.2 ± 4.4 49.0 ± 6.1 393.0 + - 66.7 CMCs 34.0 ± 2.2 * 73.0 ± 3.9 53.3 ± 4.8 408.5 ± 44.78

ALT (alanine aminotransferase) is a characteristic enzyme in cardiac muscle and hepatocyte, which increases the activity of serum in the tissue due to damage of the tissue, especially in chronic hepatitis. AST is an enzyme widely used for the diagnosis of liver disease. As the hepatocyte necrosis and hepatic tissue destruction are progressed due to the hepatic toxicity in the serum, the enzyme is released into the blood and the activity in the plasma is increased. .

As shown in Table 11 and FIG. 20A, ALT was significantly decreased in the cambium-derived stem cell (CMCs) group than in the normal control group, and showed a tendency to be lower than that of the Vitamin C group. This suggests that stem cells from cambium stem cells (CMCs) may protect the liver.

On the other hand, ALP (alkaline phosphatase) can be elevated in chronic hepatitis and cirrhosis, but it increases markedly when bile produced is not well released from hepatocytes or is blocked.

As shown in Table 11 and FIG. 20B, normal control group, Vitamin C group, and cambium-derived cambium-derived stem cells (CMCs) showed similar values.

AST (aspartate aminotransferase) is also a characteristic enzyme in the cardiac muscle and hepatocyte, which increases the activity in the serum by the damage of the tissue cell, especially in chronic hepatitis. As an enzyme widely used for the diagnosis of liver disease with ALT, the enzyme is released into the blood as the necrosis of the hepatocyte and the destruction of the liver tissue are progressed due to the hepatotoxicity in the serum, and the activity of the enzyme is increased in the plasma, .

As shown in Table 11 and FIG. 20C, it was confirmed that the number of stem cells derived from cambium-derived stem cells (CMCs) of Vitamin C group and Ginkgo biloba decreased compared with that of the normal control group. However, there was no statistical significance.

LDH (lactate dehydrogenase) is produced in large numbers in malignant tumor cells and is used to detect the presence of cancer.

As shown in Table 11 and FIG. 20D, it was confirmed that it decreased in the cambium-derived cambium-derived stem cells (CMCs) group compared to the normal control group. However, there was no statistical significance.

In the mouse experiment, the activities of antioxidant enzymes such as GSH concentration, SOD activity, catalase activity, and GPx activity were measured and ALT, AST, ALP and LDH, which are hematological indexes of liver function, were measured The results suggest that cambium stem cells (CMCs) derived from cambium have excellent antioxidative effects in vivo.

Stem cells derived from cambium CMCs ) Of the extract in vivo  Anti-inflammatory effect

When 5% dextran sodium sulfate (DSS) is mixed with drinking water for 5 to 7 days in mice, acute colitis accompanied by swelling, erythema, ulceration, and staphylococci is induced throughout the large intestine. Early lesions include stools, weight loss, and narrowing of the colon. Over time, plasma cells and lymphocytes infiltrate and develop chronic inflammation. An animal model of DSS-induced colitis has been reported to be the best model for human ulcerative colitis symptoms (Egger B, Bajaj-Elliott M, MacDonald T T, Inglin R, Eysselein VE, Buchler MW Characterization of acute murine dextran sodium sulfate colitis: cytokine profile and does dependency. Digestion 62: 240-248, 2000.). DSS-induced colitis is an inflammatory disease mainly induced by neutrophils and macrophages, and may be characterized by pro-inflammatory cytokines (IL-6, TNF-α) that induce inflammation in the mucosa. In addition, recent studies have shown that DSS-induced colitis is known to be exacerbated by Th1 cytokines secreted from T cells. Therefore, studies on the balance of Th1 type / Th2 type cytokines secreted from CD4 ⁺ effector T cells It is expected to play an important role in the treatment and prevention of inflammatory diseases. In this study, we examined the expression levels of Th1 type cytokines, IFN- and Th2 type cytokines, IL-4 and pro-inflammatory cytokines (IL-6 and TNF-α) And to demonstrate the anti-inflammatory effect of stem cells (CMCs). Sulfasalazine, which is mainly used for the treatment of ulcerative colitis, was used to compare cambium stem cells from cambium stem cells (CMCs).

9-1: Experimental animal breeding and Experimental group

Bank of cambium-derived stem cells (CMCs) of wood in For the vivo antioxidant effect, 30 female Balb / c mice of 4 weeks old were purchased from Orient (Daejeon, Korea). Experimental animals were maintained in feeder room under constant conditions (temperature: 22 ± 2 ℃, humidity: 50-60%, darkness: 12 hour period (07: 00 ~ 19: 00) After adaptation, it was used in the experiment. Six Balb / c mice were divided into 5 groups (normal control group, DSS group, DSS + SS group, DSS + CMCs group, CMCs + DSS + CMCs). Dextran sulfate sodium (5%, W / V) was added to the distilled water to induce acute colitis by free feeding for drinking water for 5 days. Thereafter, the DSS group (control group) did not receive any treatment, and the DSS + SS group (positive control group) was free to ingest sulfasalazine feed (1 mg / day / horse) for 2 weeks after inducing acute colitis. In addition, the DSS + CMCs group had free radical ingestion of cambium stem cells (CMCs) for 2 weeks after inducing acute colitis, and CMCs + DSS + CMCs group was treated with DSS before inducing acute colitis. (CMCs) feedstuffs (100 mg / day / horses) were fed for 1 week, and then the CMCs feeds of CMCs were gavaged for 2 weeks while acute colitis was induced. At this time, cambium - derived stem cells (CMCs) were prepared by mixing cambium - derived stem cells (CMCs) from lyophilized Ginkgo biloba with feed, and fed free.

9-2: Blood collection

At the end of the experiment, the blood of each group was collected from the orbital vein, left at room temperature for 30 minutes, and the blood was frozen and centrifuged at 3,000 rpm for 15 minutes at 4 ° C. The resulting supernatant (serum) was collected and stored in a -80 ° C cryocooler Respectively.

9-3: Stem cells from the cambium of cambium CMCs )end mouse On the length, condition and body weight of colon

The mice were sacrificed after cervical dislocation and the colon was cut and the length of the colon was measured and visualized by visual observation.

As shown in FIG. 21A, the length of the colon was shorter in the normal control group (56 mm) than in the DSS + SS group and the DSS + CMCs (56 mm) were similar but CMCs + DSS + CMCs (64 mm) were longer. The thickening of the colon in the DSS group was not constant compared to other groups, and the color was close to white. The fecal intensity was also the thinnest, and it was observed to be concentrated only in the center. The colonic status of the DSS + SS group and the DSS + CMCs group showed a similar tendency to that of the normal control group, with a moderate red color, and the intensity of the feces was thinner than that of the normal control group. The colonic status of the CMCs + DSS + CMCs group was brightly reddish, and the intensity of the feces was better than that of the normal control group.

As shown in FIG. 21B, the mean body weight of each experimental group was determined by CMCs + DSS + CMCs (5 days), DSS + CMCs (6 days) DSS + SS and DSS alone group (7th day) gradually increased, and the recovery of body weight was also recovered the best in CMCs + DSS + CMCs group.

9-4: Stem cells derived from the cambium of cambium CMCs ) In the colon Fecal IgA  Effect on concentration

The digestive tract is an important immune organ that not only digests and absorbs food that is a nutrient source but also eliminates harmful foreign substances such as viruses and hospital microorganisms. The gastrointestinal tract is composed of a lymphocyte of the epithelium of the intestine, lymphocytes of the lamina propria, and Payer's patch, thus forming an effective biologic defense mechanism. Immunoglobulins (Ig) are a group of proteins that function as antibodies and play an important role in the humoral immune response of the body. In particular, secretory IgA antibodies secreted from the gut mucosa epithelial cells are produced in large quantities in the small intestine and play a central role in the mucosal immune system through inhibition of absorption of harmful substances, neutralization and elimination of antigens.

Therefore, in order to measure the concentration of IgA in the intestinal feces, the colon was put into PBS solution containing protease inhibitor and finely divided and homogenized with a vortex. After centrifugation (4 ° C, 12,000 rpm, 10 minutes), the supernatant was collected and the IgA concentration in the intestinal feces was measured using IgA antibody.

First, the supernatant was allowed to react overnight at 4 ° C in a coating buffer (0.1 M carbonate, pH 9.5) on the surface of the ELISA plate. Plate was washed 5 times with 1x PBS (PBS-T) containing 0.05% Tween-20 and then blocked with 1x assay diluent solution containing 2% BSA for 2 hours at room temperature. The plate was washed three times with 1x PBS-T, and then reacted with alkaline phosphate (AP) conjugated anti-mouse Ig A for 2 hours at room temperature. Plate was washed 5 times with 1x PBS-T, and then the AP substrate solution, Phosphatase substrate, was added. The reaction rate of the plate was measured with a microplate reader at 405 nm absorbance. Mouse IgA was used as a reference material.

As a result of measuring the IgA concentration in the large intestine using an ELISA, as shown in Table 12 and FIG. 22, it was higher only in the DSS alone treatment group. This result suggests that IgA secreted by lumen by polymeric immunoglobulin receptor (pIgR) in mucosal epithelial cells is increased in the colon more than 5 times when colitis caused by DSS is induced compared to normal mouse. Damage could be predicted. In contrast, IgA secretion levels in the DSS + SS, DSS + CMCs, and CMCs + DSS + CMCs groups were almost unchanged, suggesting that mucosal tissues And that the damage was less.

Intestinal fecal IgA concentration (ng / mL) Group Colon supernatant (mean 占 SE) Normal control 46.53 ± 3.2 DSS only 304.75 ± 18.39 DSS + SS 52.67 + - 4.27 DSS + CMCs 67.5 ± 19.23 CMCs + DSS + CMCs 37.74 ± 1.07

9-5: Stem cells from cambium-derived cambium CMCs )end Mesenteric lymph node ( MLN ) Of the lymphocyte culture cytokine  Effect on concentration

Each mouse intestinal lymph node (MLN) was extracted and dissociated with collagenase solution. Monoclonal cells were separated by percoll gradient and then suspended in RPMI 1640 medium containing 10% FBS. ⁶ cells / divider as well concentration of and after the addition of Con a with 2 ㎍ / mL concentration in order to promote the proliferation of lymphocyte in _{5% CO 2 incubator 24 hr (} IFN-γ, TNF-α) or 48 hr ( IL-4, and IL-6).

The mucosal layer acts like a lubricant on the surface of the intestinal tract so that the intestinal contents can flow well inside the intestinal tract and acts as a primary line of defense when chemical, microbiological and physical damage is applied to the intestinal cells present in the lower intestine. (IL-4, IL-6, 48 hr, IFN) by DSS ingestion, because inflammatory cytokines and their mRNAs are rapidly released when intestinal microorganisms invade intestinal epithelial cells. -, TNF-a: 24 hr cultures) were examined by ELISA method to determine the effect of cambium stem cells (CMCs) derived from cambium.

First, each cytokine capture antibody was added to a 96-well ELISA plate and reacted overnight at 4 ° C. Plate was washed 5 times with 1x PBS (PBS-T) containing 0.05% Tween-20 and then blocked with 1x assay diluent solution containing 2% BSA for 2 hours at room temperature. The plate was washed three times with 1x PBS-T, and the cell culture medium or each cytokine standard protein was added thereto, followed by reaction at room temperature for 2 hours. The cells were washed 5 times with 1x PBS-T and incubated for 2 hours at room temperature. The plate was washed 5 times with 1x PBS-T, and the avidin-HRP solution was added and reacted at room temperature for 30 minutes. The reaction mixture was washed with 1x PBS-T solution 7 times, and then TMB substrate solution was added thereto. The reaction was allowed to proceed for 15 minutes at room temperature, and the reaction was stopped by adding a reaction stopping solution (H ₃ PO ₄ ). Plate reactivity was measured by absorbance at 430 nm in a microplate reader. At this time, each recombinant was used as a reference material.

As shown in Table 13 and FIG. 23, cytokine was measured in the MLN lymphocyte culture medium, which plays an important role in the mucosal immunity system using ELISA. As shown in Table 13 and FIG. 23, IFN-γ, TNF- IL-6 expression was increased. In the DSS + SS group used for the control of disease, Th2 type cytokine, Th1 type cytokine and Th1 type cytokine were detected because IL-4 expression was increased and IFN-γ, TNF-a and IL- (CMCs) from the gingko stem were more efficiently induced than those from the gingko stem-derived stem cells (CMCs) of Ginkgo biloba. The possibility of a palliative treatment was confirmed.

(Ng / mL) of IL-4, IL-6, IFN-γ and TNF-a in MLN lymphocyte culture Group IL-4
mean ± st.er IL-6
mean ± st.er IFN-y
mean ± st.er TNF-a
mean ± st.er Normal control 0.14 ± 0.01 2.17 ± 0.14 0 0.72 + 0.035 DSS only 0.025 0.005 5.72 + 0.16 3.815 ± 1.35 7.23 ± 0.07 DSS + SS 0.11 + - 0.01 1.89 0.32 0.37 + 0.25 0.301 + - 0.01 DSS + CMCs 0.087 ± 0.003 1.83 + - 0.42 0 0 CMCs + DSS + CMCs 0.158 ± 0.002 1.94 + - 0.27 0 0.269 ± 0.01

9-6: Stem cells from cambium-derived cambium CMCs ) Was serum IgA , IgG  Effect on concentration

Animals produce antibodies against a variety of antigens, including bacteria, viruses, and other foreign proteins, and defend against these antigens. Antibodies made against each antigen react with a given antigen and render them helpless. Antibodies , also called immunoglobulins, are found in the blood, saliva, and breast milk of mammals. IgA in the secretion fluid acts as the first line of defense against microbial invasion and plays an important role in preventing gastrointestinal infection.

IgA is synthesized primarily in plasma cells present in respiratory mucosa, intestinal mucosa, and glands, and some IgA enters the blood and most of it is present in or between epithelial cells. IgA between the epithelial cells is called secretory IgA (secretory IgA, sIgA) and is resistant to enzymes. In addition, IgG accounts for 75% of the antibodies in blood. IgG is the majority of bacterial antibodies, virus neutralizing antibodies, precipitating antibodies, hemagglutinins and hemolysins.

Therefore, we investigated the effect of cambium stem cells (CMCs) on the serum IgA and IgG of colitis mouse induced by DSS using ELISA method.

Serum IgA, Ig G concentration measurement (mg / mL) Group IgA
mean ± std.err IgG
mean ± std.err Normal control 4.51 + 0.498 20.72 ± 1.6 DSS only 7.29 ± 0.32 12.54 + - 1.16 DSS + SS 5.26 ± 1.07 22.59 ± 5.13 DSS + CMCs 5.58 + - 0.14 17.13 + - 4.35 CMCs + DSS + CMCs 5.70 ± 0.09 21.13 ± 1.1

As a result, as shown in Table 14 and FIG. 24A, serum IgA concentration was highest in DSS alone group, DSS + SS, DSS + Ginkgo CMCs, CMCs + DSS + CMCs group were higher than normal control group As shown in Table 14 and FIG. 24B, serum IgG concentration was lowest in DSS only group and DSS + SS, DSS + CMCs, CMCs + DSS + CMCs group were similar to normal control group.

As previously reported, colitis mice induced by DSS showed less IgG expression and increased IgA expression compared to normal. At this time, it is considered that the result of inflammation relieved the change of IgA and IgG to the normal value in the fed group of positive control SSCs and cambium stem cells derived from cambium (CMCs).

9-7: Stem cells from cambium-derived cambium CMCs ) Is spleen Lymphocyte  Effect on the cytokine concentration of culture medium

Splenocytes were isolated from each group and suspended in RPMI 1640 medium containing 10% FBS. The cells were seeded at a concentration of 2 × 10 ⁶ cells / well in a 24-well plate. To promote splenocyte proliferation, 2 μg / mL Con A was added at a concentration of 5% CO ₂ incubator for 24 hr or 48 hr.

The mucosal layer acts like a lubricant on the surface of the intestinal tract so that the intestinal contents can flow well inside the intestinal tract and acts as a primary line of defense when chemical, microbiological and physical damage is applied to the intestinal cells present in the lower intestine. (IL-4, IL-6: 48 hr, IFN-induced) by DSS ingestion, because inflammatory cytokines and their mRNAs are rapidly released when intestinal microorganisms invade intestinal epithelial cells. -, TNF-a: 24 hr cultured) were examined by ELISA method to determine the effect of cambium stem cells (CMCs) derived from cambium.

Measurement of IL-4, IL-6, IFN-γ and TNF-a concentrations in spleen lymphocyte culture (ng / mL) Group IL-4
mean ± st.er IL-6
mean ± st.er IFN-y
mean ± st.er TNF-a
mean ± st.er Normal control 0.66 + 0.04 0 0 0 DSS only 0.086 0.03 0.61 + 0.02 4.0 ± 0.08 0.087 ± 0.01 DSS + SS 1.705 + 0.12 0.89 + 0.07 3.0 ± 0.14 0.07 ± 0.004 DSS + CMCs 3.23 ± 0.011 0.41 + 0.04 0.77 ± 0.15 0 CMCs + DSS + CMCs 1.58 ± 0.11 1.54 + 0.06 0.84 0.06 0.059 ± 0.01

In order to examine the effect of Th1 type T cells on DSS-induced colites in the whole body immunity, each cytokine was examined from the spleen lymphocyte culture medium. As shown in Table 15 and FIG. 25, (CMCs) in the SS or Ginkgo biloba showed a significant increase in Th1-type cytokine and pro-inflammatory cytokine compared with the Th1-type cytokine The inflammatory response is expected to be mitigated by the decrease in the expression level of pro-inflammatory cytokine.

In using the cambium-derived stem cells (CMCs) of the ginkgo tree colitis mouse induced by DSS vivo The anti-inflammatory effect (visual observation of colon tissue, IgA concentration in intestine, cytokine measurement in MLN lymphocyte culture, measurement of IgA and IgG concentration in serum, cytokine measurement in spleen lymphocyte culture, etc.) And it was found.

Manufacturing example  One. Pharmaceutical preparation Manufacturing example

Formulation example  1-1: Preparation of tablets

100 mg of the stem cell extract prepared in Example 2 was prepared by mixing 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate according to a conventional purification method.

Formulation example  1-2: Preparation of capsules

500 mg of the stem cell extract prepared in Example 2 was filled in soft gelatin capsules to prepare capsules.

Formulation example  1-3: Preparation of Syrup

100 mL of a syrup was prepared according to a conventional method for preparing a liquid preparation, based on 1 g of the stem cells obtained in Example 1, 10 g of isomerized sugar, 5 g of mannitol, and an appropriate amount of purified water.

Formulation example  1-4: Preparation of injection

200 mg of the stem cell extract prepared in Example 2 was dissolved in 200 mg of physiological saline containing polyoxyethylene hydrogenated castor oil under heating to prepare an injection containing the mixed extract at a concentration of 0.1%.

Manufacturing example  2. Manufacture of functional food

Manufacturing example  2-1: Preparation of functional beverage

200 mg of the stem cells obtained in Example 1 were dissolved in 96 ml of water, 500 mg of vitamin C as an adjuvant, 1 g of citric acid and oligosaccharide as a mating agent were added, 0.05 g of sodium benzoate was added thereto, and purified water To make a total volume of 100 ml to prepare a functional beverage.

Manufacturing example  2-2: Production of functional beverage

After 200 mg of the stem cell extract prepared in Example 2 was dissolved in 96 mL of water, 500 mg of vitamin C as an adjuvant, 1 g of citric acid and oligosaccharide as a mating agent were added, and 0.05 g of sodium benzoate was added thereto as a beverage. To make 100 mL of the total amount.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> UNHWA CORPORATION <120> Anti-inflammatory composition comprising plant stem cell derived          from cambium of family ginkgoaceae <130> P10-B315 <160> 6 <170> Kopatentin 1.71 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> COX-2 gene Forward primer <400> 1 aagaagaaag ttcattcctg atccc 25 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> COX-2 gene Reverse primer <400> 2 tgactgtggg aggatacatc tctcc 25 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> iNOS gene Forward primer <400> 3 ctgcagcact tggatcagga acctg 25 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> iNOS gene Reverse primer <400> 4 gggagtagcc tgtgtgcacc tggaa 25 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH gene Forward primer <400> 5 ccgatgcccc catgtttgtg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH gene Reverse primer <400> 6 ggccatgcca gtgagcttcc 20

Claims (22)

An anti-inflammatory composition comprising at least one of cambium-derived stem cells derived from Ginkgo biloba cambium, an extract thereof, and a culture thereof, which is a native undifferentiated cell derived from a cambium (Ginkgo biloba) cambium.
The anti-inflammatory composition according to claim 1, wherein the cambium stem cell derived from cambium has at least one of the following characteristics:
(a) contains a large number of single cells or a small-sized cell aggregate as compared to the dedifferentiated callus of ginkgo in suspension culture;
(b) exhibits morphological features with multiple vacuoles;
(c) has an increased activity of mitochondria compared to demineralized callus of ginkgo;
(d) Growth rate is faster and longer than ginkgo's demineralized callus; And
(e) Has low sensitivity to shear stress in bioreactors as compared to demineralized callus of ginkgo.
3. The anti-inflammatory composition according to claim 2, wherein the cambium-derived cambium stem cells have at least two of the characteristics (a) to (e).
The anti-inflammatory composition according to claim 2, wherein the cambium-derived cambium-derived stem cells have at least three of the characteristics (a) to (e).
[3] The anti-inflammatory composition according to claim 2, wherein the cambium-derived cambium-derived stem cells have at least four characteristics of (a) to (e).
The anti-inflammatory composition according to claim 2, wherein the cambium-derived cambium-derived stem cells have the characteristics (a) to (e).
The anti-inflammatory composition according to claim 1, wherein the cambium-derived cambium-derived stem cells are separated by a separation method comprising the steps of:
(a) obtaining a cambium-containing tissue from ginkgo;
(b) culturing the obtained cambium-containing tissue in a medium; And
(c) obtaining cambium-derived stem cells from cambium separated from calli of said cultured tissue.
2. The method of claim 1, wherein the culture comprises contacting the stem cells with 3-5% (g / L) raw sugar or sugar; And methyl jasmonate. &Lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt;
The anti-inflammatory composition according to claim 1, wherein the extract is extracted using a solvent selected from the group consisting of distilled water, alcohol, acetone, dimethyl sulfoxide (DMSO), and a mixed solvent thereof.
delete delete A functional food for preventing or ameliorating inflammation, which contains at least one of cambium-derived stem cells derived from Ginkgo biloba cambium derived from Ginkgo biloba cambium, undifferentiated undifferentiated cells, extract thereof and cultures thereof.
13. The functional food according to claim 12, wherein the cambium stem cell derived from cambium has at least one of the following characteristics:
(a) contains a large number of single cells or a small-sized cell aggregate as compared to the dedifferentiated callus of ginkgo in suspension culture;
(b) exhibits morphological features with multiple vacuoles;
(c) has an increased activity of mitochondria compared to demineralized callus of ginkgo;
(d) Growth rate is faster and longer than ginkgo's demineralized callus; And
(e) Has low sensitivity to shear stress in bioreactors as compared to demineralized callus of ginkgo.
14. The functional food according to claim 13, wherein the cambium-derived cambium stem cell has at least two of the characteristics (a) to (e).
14. The functional food according to claim 13, wherein the cambium-derived cambium-derived stem cells have at least three of the characteristics (a) to (e).
14. The functional food according to claim 13, wherein the cambium-derived cambium-derived stem cells have at least four characteristics of (a) to (e).
14. The functional food according to claim 13, wherein the cambium stem cell derived from cambium has the characteristics (a) to (e).
13. The functional food according to claim 12, wherein the cambium-derived cambium-derived stem cells are separated by a separation method comprising the steps of:
(a) obtaining a cambium-containing tissue from ginkgo;
(b) culturing the obtained cambium-containing tissue in a medium; And
(c) obtaining cambium-derived stem cells from cambium separated from calli of said cultured tissue.
13. The method according to claim 12, wherein the culture comprises culturing the stem cells in a medium containing 3-5% (g / L) raw sugar or sugar; And methyl jasmonate. The functional food according to claim 1 or 2,
13. The functional food according to claim 12, wherein the extract is extracted using a solvent selected from the group consisting of distilled water, alcohol, acetone, dimethyl sulfoxide (DMSO), and a mixed solvent thereof.
delete delete

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