KR20120057370A - Anti-aging Composition Comprising Plant Stem Cell Derived from Cambium of Family Ginkgoaceae - Google Patents

Abstract

PURPOSE: An anti-oxidative or anti-aging composition containing Ginkgoaceae cambium-derived stem cells or extract, crushed material, or culture thereof is provided to suppress active oxygen. CONSTITUTION: An anti-aging composition contains Ginkgoaceae cambium-derived stem cells or extract, pulverized material, or culture thereof. A method for isolating the stem cells comprises: a step of preparing cambium-containing tissue from Ginkgoaceae; a step of culturing the tissue in a medium; and a step of isolating cells from the cambium using distilled water, alcohol, acetone, DMSO(dimethyl sulfoxide) or mixture solvent thereof. A cosmetic composition for anti-aging or whitening contains the stem cells, or extract, pulverized material, or culture thereof.

Description

Anti-aging Composition Comprising Plant Stem Cell Derived from Cambium of Family Ginkgoaceae}

The present invention relates to a composition for anti-aging containing any one or more of the stem cells derived from the cambium of the Ginkgoaceae, its extract, its lysate and its culture.

Aging is a functional, structural, and biochemical process that occurs continuously from birth to death, and occurs throughout the cells and tissues that make up the human body. It shows metabolic rate, increased disease, and decreased adaptability. This is a phenomenon that leads to the death of the whole body. Theories explaining the aging process and causes are largely genetic (Chung.HY et. al ., Kor . J. Gerontol . , 2: 1, 1992) and consumption theory (Chung . HY et al . , Kor . J. Gerontol ., 2: 1, 1992). Consumption theory explains that the constituent cells of an organism deteriorate with time (wear & tear theory) or by accumulation of harmful substances, in particular human metabolism, radiation exposure, viruses and heavy metals. The free radical theory that the free radicals generated through air pollution and the like form a highly toxic substance, not only promotes aging but also causes various diseases related to aging.

Free radicals collectively refer to a material having an electron that is not paired with the outermost electron orbit, and its structure is very unstable and highly reactive due to its nature of obtaining and stabilizing electrons. In particular, free radicals derived from oxygen are called free radicals, and they react with proteins, lipids, and carbohydrates, causing lipid peroxidation, DNA damage, and oxidation of proteins, resulting in damage to cellular structures, resulting in cell death. In particular, it is involved in the inflammatory process as a cause of vascular aging and increases homocysteine in arteriosclerosis, Alzheimer's, and blood.

Oxygen-related toxic substances in humans are called reactive oxygen species (ROS). These types of ROS include superoxide, hydroxyl, peroxyl, alkoxyl, Free radicals such as hydroperoxyl and hydrogen peroxide, hypochlorous acid, ozone, singlet oxygen, and peroxynit There are non free radicals, such as peroxinitrite. The most studied and important role of oxygen toxicity is superoxide free radical (active oxygen or harmful oxygen) is known (Fridovich L., Science , 201: 175,1978).

ROS generated in the mitochondria of cells during aging is a target for oxidative damage (Lesnefsky, EJ & Hoppel, CL Arch . Biochem . Biophys . 420, 287, 2003). This free radical theory, an intracellular oxygen radical, has been reported to correlate with the oxidative stress associated with aging and the resulting aging-related diseases (Finkel, T. & Holbrook, NJ, Science 408; 239, It is reported that these oxidative stresses play an important role in inducing senescence (Chen Q. et. al ., Proc . Natl . Acad . Sci . USA May 9; 92 (10): 4337, 1995; Packer L. & Fuehr K., Nature , 267 (5610): 423, 1977). This oxidative stress is a term used to describe the damage to cells caused by the oxidation of macromolecules by increasing levels of reactive oxygen species and reduced antioxidant reserves (Thomas C. & Squier, Experimental). Gerontology , 36; 1539, 2001).

Aged cells undergoing replicative senescence may produce higher levels of ROS than young cells and may produce toxic byproducts during normal metabolic processes such as superoxide, hydrogen peroxide, hygisyl radicals, etc. do. Experiments with older test animals indicate that tissues accumulate oxidative damage to their DNA, proteins, and lipids (Chen, Ann . NY Acad . Sci . , 908: 111, 2000). ROS and, in particular, high hydroxyls, attack DNA and cause the formation of 8-oxo-2'-deoxyguanosine and other potent mutagenic products, with the rate of ROS associated with each species associated with life-span, which is associated with varying rates of aging and It is a decisive factor in the definition of diseases associated with aging (Finkel & Holbrook, Nature , 408 (6809): 239, 2000).

"In young cultured cells, replication capacity gradually decreases as the number of divisions progresses, giving doubling limited, which eventually loses its ability to proliferate and ends growth," first presented by Hayflick and Moorhead in 1961. ROS are human fibroblasts in As an experimental model for in vitro aging and intracellular aging, it has been used to define the molecular changes associated with aging of human cells (Hayflick L. & Moorhead PS, Exp . Cell) . Res ., 25: 585, 1961).

All aerobic organisms, including humans, are equipped with self-defense mechanisms to protect against free radicals that always occur during oxygen-based energy metabolism, but the production of free radicals beyond the defenses of tissues has recently been called adult disease. In addition to arthritis and circulatory disorders, it is the cause of many diseases such as dementia (Halliwell et. al ., Drugs , 42: 569, 1991; Fukuzawa et al ., J. Act . oxyg . Free Rad., 1:55, 1990).

Often free radical la called the active oxygen is the most stable form of oxygen which singlet oxygen (3O ₂₎ is a superoxide ion singlet oxygen that is generated is reduced in oxidation-reduction process (Superoxide anion; O ^{2 -)} and hydrogen peroxide (H ₂ O ₂ ), free radicals in unpaired states such as hydroxy radicals (? OH), which cause diseases by damaging factors of the immune system such as proteins, DNA, enzymes and T cells (Regnstrom et al. al ., Lancet . , 16: 1183, 1992; Gey et al ., Am . Ac . J. Cin . Nutr ., 53: 326, 1991).

For this reason, researches on the development of antioxidants have been actively conducted, and antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase, which are enzyme-protective antioxidants, and vitamins that are natural antioxidants E, vitamin C, carotenoids, glutathione, and synthetic antioxidant butylhydroxyanisole (BHA) dibutylhydroxytoluene (3,5- (t-Butyl)- Many antioxidants, such as 4-hydroxytoluene (BHT), are known, but antioxidant enzymes decrease their protection against free radicals as they age, while synthetic antioxidants are safer and more potent due to their mutagenicity and toxicity. There is an urgent need for the development of natural antioxidants (Hatano et. al ., Natural Medicines , 49: 359, 1995; Masaki et al ., Biol . Pharm . Bull , 18: 162, 1995).

Therefore, the present inventors have made intensive efforts to develop a natural-derived composition excellent in anti-aging activity, and confirmed that the stem cells derived from the cambium of the ginkgo biloba and the culture medium of ginkgo biloba have an excellent inhibitory effect on aging and have a whitening activity. The present invention has been completed.

It is an object of the present invention to provide a composition derived from natural products exhibiting anti-aging and delaying effects.

In order to achieve the above object, the present invention is derived from the formation layer of ginkgo biloba, the stem cell derived from the formation layer of ginkgo biloba, which is a congenital undifferentiated cell without dedifferentiation, its extracts, its lysate and its culture It provides an anti-aging composition containing.

The present invention also provides an anti-aging cosmetic composition containing any one or more of the stem cells derived from the cambium of the Ginkgoaceae, its extract, its lysate and its culture.

The present invention also provides an anti-aging functional food containing any one or more of the stem cells derived from the cambium of the Ginkgoaceae, its extract, its lysate and its culture.

The present invention also provides a cosmetic composition for whitening containing any one or more of the stem cells derived from the cambium of the Ginkgoaceae, its extract, its lysate and its culture.

The present invention has the effect of providing an anti-aging composition containing any one or more of the stem cells derived from the cambium of the Ginkgoaceae, its extracts, its lysate and its culture as an active ingredient. The composition according to the present invention inhibits the production of enzymes associated with the aging process with better efficiency than the retinoic acid known to have a strong anti-wrinkle effect, and has an effect of inhibiting free radicals that are increased by UV irradiation, thereby preventing and delaying aging. Useful for In addition, it has a melanin formation inhibitory effect, it is also useful as a cosmetic composition for whitening.

Figure 1 is a photograph of the formation layer in the cross section of the material plant (Ginkgo biloba).
Figure 2 is a picture of the induction and separation of stem cells according to the present invention, 2A is a picture showing an extremely different form of stem cells (arrows) and phloem cells (star), 2B is cultured after separating the stem cells 3 weeks later (scale bar: 1 mm).
Figure 3 is a photograph of the solid culture of stem cells (A) and bark sections derived callus (B) of Ginkgo biloba according to the present invention (scale bar: 0.25 mm).
Figure 4 is a micrograph observing the degree of cell aggregation of the callus (B) induced in the stem cells (A) and the bark tissue of ginkgo biloba of the present culture (x100, scale bar: 50 ㎛).
5 is a micrograph of a stem cell (B) and a ginkgo somatic cell (B) according to the present invention. The scale bar at the bottom left is 15 μm.
Figure 6 is a micrograph of the stem cells (A) and the callus (B) derived from the ginkgo bark tissue according to the present invention observed after staining the vacuole using Neutral red, the scale bar of the lower right is 25 ㎛.
Figure 7 is a photograph of the mitochondria of the stem cells (A) and callus (B) derived from the ginkgo bark tissue according to the present invention, the scale bar at the bottom left is 15 ㎛.
8 is a graph showing the cell growth curve of the stem cell bioreactor according to the present invention.
Figure 9 is a graph showing the growth rate ratio of the callus (callus) induced in stem cells (stem cell) and ginkgo bark tissue according to the present invention.
FIG. 10 is a photograph showing a scale-up process in a 3 L air-lift bioreactor (A), a 20 L air-lift bioreactor (B) and a 250 L air-lift bioreactor (C).
11 is a graph showing the cell survival rate after cryopreservation of callus induced in stem cells and ginkgo bark tissues according to the present invention.
Figure 12 is a graph showing the results of measuring the survival rate of the human keratinocytes of the stem cell extract and culture solution according to the present invention.
13 is a graph showing the MMP-1 production inhibitory effect of the stem cell extract and the culture medium according to the present invention when inducing MMP-1 by ultraviolet irradiation to fibroblasts.
14 is a graph showing the active oxygen production inhibitory effect of the stem cell extract according to the present invention when inducing free radicals by irradiating UV light to human keratinocyte line.
15 is a graph showing the active oxygen production inhibitory effect of the stem cell culture according to the present invention when inducing free radicals by irradiating UV light to human keratinocyte line.
16 is a graph showing the results of measuring the melanin formation inhibitory activity when the stem cell extract and the culture solution according to the present invention.

Unless defined otherwise, 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.

Definitions of main terms used in the detailed description of the present invention are as follows.

Herein, the vascular "forming layer" is located in the stems and roots as a lateral meristem located in the vascular tissue of a plant. The activity of the cambium causes hypertrophy of the plant, resulting in the presence of large vegetation with more than 11,000 years of age. Genetically speaking, the vascular forming layer originates from the procambium, so that it can be conveniently divided into gradually divided mitotic tissues while maintaining mitotic continuity. In the present invention, the formation layer is interpreted to include a formation layer. It is obvious that such a formation layer and a typical formation layer are the same primary fission structure, and the same effect will be acquired by using a formation layer and a typical formation structure in this invention.

As used herein, "cracked material" refers to a cell lysate obtained by crushing a cell by a chemical method or a physical method using a detergent or the like, and "extract" of a cell line is a material obtained by dissolving the cell in a solvent and using distillation or evaporation. Can be concentrated. In addition, the cell line "culture medium" means a cell culture solution remaining after culturing the cells, excluding the cells. Additionally, as used herein, the term "culture" is a substance containing a culture medium and / or a cultured cell line, wherein the cultured cell line is a concept including all cell lines that are differentiated by culture conditions or have improved production and / or secretion capacity of useful materials. .

Plant “stem cells” as used herein refer to innate undifferentiated cells that are genetically more stable without undergoing dedifferentiation.

As used herein, "innately undifferentiated" refers to maintaining a pre-differentiation state, rather than being present in an undifferentiated state through a dedifferentiation process.

As used herein, "callus" refers to a cell or cell mass that has not been differentiated through a dedifferentiation process ( Proc . Natl . Acad . Sci . USA , 99 (25): 15843, 2002).

In one aspect, the present invention relates to a composition for anti-aging containing any one or more of the stem cells derived from the cambium of the Ginkgoaceae, its extract, its lysate and its culture.

Stem cells derived from the cambium of the Ginkgoaceae according to the present invention is derived from the cambium's cambium, and are innate undifferentiated cells. It may be characterized by having at least one of the following characteristics:

(a) contains a large number of single cells or a small size of cell aggregates in suspension culture compared to the dedifferentiated callus of Ginkgo biloba;

(b) exhibit morphological features with multiple vacuoles;

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

(d) can grow faster and grow longer than de-differentiated callus of Ginkgo biloba; And

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

In this case, "having a large number of vacuoles" means having a plurality of vacuoles two or more times as compared with cells derived from dedifferentiated callus of Ginkgo biloba. In addition, the stem cells according to the present invention has a small vacuole in size compared with the cells derived from the callus of the ginkgo family. In addition, having "a large number of advanced forms of mitochondria" means having more than a two-fold majority of mitochondria that are actively moving under light microscope BX41 (Olympus, Japan) as compared to cells derived from dedifferentiated callus of Ginkgo biloba. .

In the present invention, the stem cells may be characterized by having at least two or more of the characteristics of (a) to (e), preferably at least three or more of the characteristics of (a) to (e) It may be characterized by having a characteristic, and more preferably at least four or more of the characteristics of the above (a) to (e). In addition, in the present invention, the stem cells may be characterized by having all the properties of (a) to (e).

On the other hand, the stem cells according to the present invention comprises the steps of (a) obtaining a cambium-containing tissue from the Ginkgoaceae; (b) culturing the obtained cambium-containing tissue in a medium; And (c) can be obtained by a separation method comprising the step of obtaining a cambium-derived stem cells by separating the cells from the cambium, wherein step (b) is a cambium layer to grow from the cambium layer by culturing the cambium-containing tissues It may be characterized by inducing a layer of (cambium layer), the step (c) may be characterized in that to obtain a cambium-derived stem cells by separating the cambium layer of the cambium layer. In addition, the step (b) may be characterized by culturing in a medium containing auxin (auxin), wherein as the auxin, NAA (α-naphtalene acetic acid), IAA (indole-3-acetic acid) or pickle Rolam (picloram) can be used, such auxin may be characterized in that it is included in a concentration of 1 ~ 5 mg / L. In addition, the step (c) may be characterized in that to form a stem cell derived from the cambium layer by separating the cambium layer of the cambium layer from the callus layer that is amorphously proliferated from the portion other than the cambium.

The culture in the present invention, the stem cells as an eliminator 3 to 5% (g / L) of raw sugar or sugar; Or further culturing in a medium containing any one or more of methyl jasmonate, chitosan, phenylalanine, benzoic acid, ABA, salicylic acid, and sodium acetate. It can be characterized in that obtained by performing. At this time, preferably the medium is 3 to 5% (g / L) raw sugar or sugar; And methyl jasmonate, fungal extract, bacterial extract, yeast extract, chitosan, glucomanan, glucan, phenylalanine, benzoic acid, salicylic acid ), Arachidonic acid, STS, mevalonalonate N-benzolyglycine, ABA, SNP, IPP, BHT, CCC, ethephon, hipuic acid, ceric ammonium nitrate, 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 It may be characterized in that the medium containing.

In the present invention, it is also possible to use a culture obtained by applying physical and chemical stress to the stem cells by treating ultraviolet rays, heat, ethylene, antifungal agents, antibiotics, heavy metal salts and high salt concentrations as an eliminator.

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

In one embodiment of the present invention Ginkgo ( Ginkgo ) of the Ginkgoaceae genus Ginkgo biloba ) to determine the anti-aging effect by separating the stem cells from the cambium-derived layer of Ginkgo biloba , the present invention preferably uses the cambium-derived stem cells of the genus Ginkgo plant, more preferably It may be characterized by using a cambium-derived stem cell.

In the embodiment of the present invention, it was confirmed that the stem cell extract according to the present invention has the effect of removing the active oxygen species generated by ultraviolet irradiation.

Therefore, the stem cell extract and its culture according to the present invention can also be usefully used as a very excellent antioxidant composition. In addition, antioxidant activity such as removal of reactive oxygen species has the effect of preventing aging.

In addition, in one embodiment of the present invention, the inhibitory rate of production of type I collagenase (matrix metalloproteinase-1, MMP-1) induced by ultraviolet irradiation for the stem cell extract and its culture according to the present invention was measured, As a result, it was confirmed that both the stem cell extract and the culture medium had superior MMP-1 production inhibitory effect than retinoic acid, which is known to have an anti wrinkle effect. In particular, it was confirmed that the stem cell extract and the culture medium has a much lower inhibitory effect of MMP-1 expression inhibition effect than before UV irradiation.

Therefore, the stem cell extract and its culture according to the present invention can be usefully used as a very good anti-aging composition.

Thus, in the present invention, even if there is no specific embodiment suggesting that the composition containing the stem cells exhibit anti-aging activity, as confirmed above, it was confirmed that the stem cell extract has anti-aging activity, according to the present invention It will be apparent to those skilled in the art that even in the case of the stem cell itself or a composition containing the lysate, anti-aging activity can be prevented to prevent aging.

An anti-aging composition containing any one or more of the stem cells, extracts thereof, lysates and cultures thereof according to the present invention as an active ingredient, each alone or at least one pharmaceutically acceptable carrier, excipient or diluent It may be provided as a pharmaceutical composition, the stem cells, etc. may be included in the pharmaceutical composition in an appropriate pharmaceutically effective amount according to the disease and its severity, the age, weight, health status, sex, route of administration and treatment period of the patient. have.

As used herein, "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and that, when administered to a human, typically does not cause an allergic reaction such as gastrointestinal disorders, dizziness, or the like. Examples of such carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition may further include fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives. Pharmaceutical compositions of the invention can also be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders.

On the other hand, the stem cell extract according to the present invention and its culture medium showed a suppressive effect of MMP-1 production of collagenase to a superior degree compared to retinoic acid, which is known to be excellent skin anti-aging effect, stem according to the present invention Since the composition comprising the cells, the extract, the lysate or the culture medium has an effect of inhibiting the degradation of collagen, it has been shown to have an anti-aging and anti-wrinkle effect, and thus it was found to be very useful as an anti-aging cosmetic composition. Therefore, in another aspect, the present invention relates to an anti-aging cosmetic composition containing any one or more of the stem cells derived from the cambium-derived layer of ginkgo biloba, its extract, its lysate and its culture as an active ingredient.

In one embodiment of the present invention, it was confirmed that the stem cell extract and the culture medium according to the present invention has an effect of inhibiting the melanin forming activity. This melanin formation inhibitory effect has a whitening effect. Accordingly, the present invention is, in another aspect, a whitening derived from the cambium of the ginkgo family and containing any one or more of the stem cells derived from the cambium of the ginkgo family, which is a congenital undifferentiated cell without dedifferentiation, its extract, its lysate and its culture. It relates to a cosmetic composition for.

In addition, in another embodiment of the present invention, it was confirmed that the stem cell extract and the culture medium according to the present invention has an effect of inhibiting melanin forming activity. This melanin formation inhibitory effect has a whitening effect. Accordingly, the present invention is, in another aspect, a whitening derived from the cambium of the ginkgo family and containing any one or more of the stem cells derived from the cambium of the ginkgo family, which is a congenital undifferentiated cell without dedifferentiation, its extract, its lysate and its culture. It relates to a cosmetic composition for.

Thus, in the present invention, even if there is no specific embodiment suggesting that the composition containing the stem cells show whitening activity, as described above, it was confirmed that the stem cell extract has a whitening activity, the stem cells according to the present invention It will be apparent to those skilled in the art that even in the case of a composition containing itself or its lysate, a whitening effect is obtained through melanin formation inhibition.

The components included in the cosmetic composition of the present invention include components conventionally used in cosmetic compositions in addition to the stem cells and the like as active ingredients, and include, for example, conventional solvents, stabilizers, solubilizers, emulsifiers, vitamins, pigments and flavors. Adjuvants, and carriers.

The cosmetic composition of the present invention may be prepared in any formulation commonly prepared in the art, but preferably, lotion, nutrition lotion, nutrition cream, massage cream, nutrition essence, pack, makeup base, foundation, body oil , Hair oil, shampoo, rinse is characterized in that it is prepared in a formulation selected from the group consisting of.

Regardless of the formulation of the cosmetic composition, any one or more of the stem cells of the present invention, its extracts, its lysates, and its cultures may have antioxidant and inhibitory effects on melanin formation, thereby eliminating free radicals in the skin. It has a protective effect on the antioxidant system of cells, which can prevent and delay skin aging caused by oxidation by free radicals.It can prevent skin aging and improve wrinkles by suppressing MMP-1 production. It can have a whitening effect.

In addition, the present invention relates to an anti-aging functional food containing any one or more of the stem cells, extracts thereof, lysates and cultures thereof according to the present invention as an active ingredient.

As used herein, the term "functional food" means that the functionality of the food is improved by adding any one or more of the stem cells or extracts thereof, lysates, and cultures thereof according to the present invention.

Example

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

In particular, the following examples confirmed the anti-aging and whitening effect of the cambium-derived stem cell extract and its culture, but it is possible to obtain similar results using the stem cells themselves, having ordinary skill in the art. It will be obvious to him.

Obtaining Stem Cells Derived from the Glial Layer of Ginkgo Biloba

1-1: Preparation of Plant Materials

Ginkgo and Ginkgo ( Ginkgo) biloba , Buyeo, Korea , Chungcheongnam-do), as shown in Figure 1, through the phloroglucinol staining can observe the xylem and phloem fibers, it can be seen that the formation layer (cambium) between them. In order to obtain the cambium-derived stem cells from these ginkgo biloba, the stems of the ginkgo biloba were first harvested and then immediately deposited in 100 mg / L ascorbic acid (L-ascorbic acid, DUCHEFA, The Netherlands) solution for transportation. Stored. At this time, in order to obtain the same stem cells using the typical layer, the twigs are collected instead of the stems.

Then, 1% benomyl (Dongbu Hannong Chemical, Korea), 1% daconil (Dongbu Hannong Chemical, Korea), 1% streptomycin (sterptomycin sulphate, DUCHEFA, The Netherlands), 0.1% cefotaxime sodium After 24 hours of pretreatment in a mixed solution of DUCHEFA, The Netherlands), the mixture was washed with tap water for 30 minutes to remove phenolic compounds and residual chemicals. After 1 minute in 70% ethanol, 15 minutes in 30% hydrogen peroxide (LG Chemical, Korea), 15 minutes in 1% CLOROX solution, 5 minutes in 3% CLOROX solution Washed 3-4 times.

1-2: Formation layer from stem Intersecting  Manufacturing Organization

When the bark tissue containing the formation layer of the stem (twig when using the typical layer), which had undergone the sterilization process of Example 1-1, was lifted, it was easily peeled off the xylem. Peeled tissues constitute sections that include the formation layer, phloem, cortex, and epidermis.

1-3: Stem cell induction step of the cambium layer

The fragment comprising the formation layer prepared in Example 1-2 was cultured by denture on the stem cell induction medium (media 1) of Table 1.

Cell line induced 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

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

Cell division was observed visually from the formation layer on the 4th to 7th days of the initial culture, and after 15 days of culture, amorphous callus began to be induced by dedifferentiation from the layer consisting of the dead skin layer and the epidermis. After 30 days of incubation, the cultured cambium began to separate from the layer containing the dead, that is, the amorphous callus layer (FIG. 2A: stem cells (arrows), pholem cells (asterisk), and the two layers naturally separated. Wait until the complete separation was separated and incubated in different petridish (petridish). After separation, the white and soft parts with good growth rate were passaged every 14 days with the same fresh medium as the induced medium. Figure 2B is a photograph after incubation for 3 weeks by separating the cambium-derived stem cells.

Meanwhile, bark sections of Ginkgo biloba were sterilized for comparison and then cultured in the medium of Table 1. As a result, as shown in FIG. 3B, bark sections were callus by dedifferentiation. It can be observed that Callus derived from bark sections was amorphous due to the difference in the rate of division between cells, such as tetrahedral tissue, and showed unstable growth rate and browning tendency. Callus derived from browned and agglomerated bark fragments eventually became dull by the phenolic compounds they secreted and eventually became necrotic. That is, callus derived from bark from six months later was difficult to maintain and incubate. On the other hand, as shown in Figure 3A, the cambium-derived stem cells were stably maintained without change in cell growth rate, growth pattern, and degree of aggregation during long-term culture, thereby enabling mass culture.

1-4: Proliferation and Characterization of Stem Cells Derived from the Formation Layer of Ginkgo Biloba

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

Suspension medium 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

When examining the degree of cell aggregation under the optical microscope BX41 (Olympus, Japan), the stem cells according to the present invention, as shown in Figure 4A, contains a large number of single cells in suspension culture, some of the small cell aggregates It was confirmed that the presence, but in the case of somatic cells (callus derived from the bark tissue) of the control ginkgo, it was observed that the aggregation as shown in Figure 4B.

On the other hand, as shown in Figure 5A, stem cells according to the present invention was able to observe the morphological characteristics having a plurality of vacuole (vacuole). This feature is a feature that appears due to causes such as pressure in the undifferentiated cells present in the plant, it was confirmed that the stem cells according to the present invention is in an undifferentiated state. In the case of somatic cells (callus derived from bark tissue) of ordinary ginkgo biloba, these characteristics could not be confirmed, as shown in FIG. 5B. To examine this in more detail, as a result of staining the vacuole with neutral red, stem cells according to the present invention were able to identify a number of small vacuoles of red as shown in FIG. 6A, and the somatic cells of the common ginkgo (bark) In the case of callus induced in the tissue, as shown in FIG. 6B, one large central vacuole was found.

On the other hand, stem cells according to the present invention was observed through optical microscope BX41 (Olympus, Japan), as a result, it was able to observe a large number of mitochondria very active in terms of movement. 7A shows that stem cells according to the present invention have a plurality of mitochondria, and arrows indicate mitochondria. In contrast, in the case of somatic cells (callus derived from the bark tissue) of the general ginkgo tree, as shown in FIG. 7B, such characteristics could not be confirmed.

On the other hand, in order to measure the growth rate according to the culture period, as a result of measuring the growth rate of the cell culture using an air-lift bioreactor (airlift bioreactor, Samsung Science, Korea) having a volume of 3L, as shown in Figure 8, Growth rate of 2 times in 1 week culture and 3 times in 2 weeks culture was confirmed. At this time, the medium was used in the liquid medium of Table 2, it was kept constant at 25 ± 1 ℃ under dark conditions. In addition, the same medium and conditions were compared with each other by culturing stem cells derived from the cambium-derived layer of the ginkgo biloba according to the present invention in a 3L air-lift bioreactor.

Growth rate and growth index (GI) measurement results 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 induced in the ginkgo bark tissue was 2.3 times, and the growth index (GI) (growth index = (maximum DCW-initial DCW) / initial DCW) Although only 1.3, 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 were higher than those of callus derived from ginkgo bark tissue. In the case of reactors, cell viability is rapidly reduced due to the formation of growth rings in the reactor and the cohesion of plant cultures in the culture and the rigidity of the cell walls, and the sensitivity to shear stress. The growth of the ring in the bioreactor was made very small, and a simple stimulation to the incubator to move the medium, the ring of the inner wall was easily resolved. In addition, the aggregation is small and has many vacuoles, so the sensitivity to shear stress is weak, so that the cell viability is hardly reduced.

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, the result of incubation for 10 days in a 3 L air flotation bioreactor, a 20 L air flotation bioreactor and a 250 L air flotation bioreactor, mass culture as 250 L Proliferation was also observed, and the amount of dry cells per liter was increased during the 250 L mass culture.

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

On the other hand, cryopreservation was performed on the callus derived from the ginkgo bark tissue and the stem cells derived from the ginkgo biloba forming layer according to the present invention. Suspension cultures are used for 6 to 8 days of culture, and cryopreservatives contain 0.5 M glycerol (DUCHEFA, The Netherlands) and 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 cell inoculation treated with cryopreservative is 200 mg / mL. The cryopreservative treated suspension cells were kept in a freezer for 30 minutes and then stored in a deep freezer for 3 hours and then frozen by immersion in liquid nitrogen.

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

Results of survival rate measurement 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 Figure 11 and Table 5, the callus derived from the ginkgo bark tissue was confirmed to show a very low survival rate compared to the stem cells derived from the ginkgo biloba forming layer of the present invention.

In addition, the characteristics that characterize the plant stem cells have a pluripotency in addition to self renewal (self renewal). In order to induce the differentiation of stem cells derived from ginkgo biloba-derived stem cells, it was confirmed that the conduit elements were differentiated from the cambium-derived stem cells when cultured at 25 ± 1 ° C. under dark conditions in growth medium containing MS. .

1-5: Elisitarian  process

Stem cells suspended in culture for 14 days as in Example 1-4 were collected in sterile water for 14 days and cancer cells were cultured for 14 days in medium containing 100 μM of methyl jasmonate. The following experiment was performed.

Preparation of Extracts from Stem Cells Derived from the Gypsum Formation of Ginkgo

2-1: DMSO  ( dimethyl sulfoxide ) Preparation of extract

(Iii) 500 mL of DMSO was added to 500 g of the stem cells isolated in Example 1 from which the culture solution was removed, and dissolved by stirring at 50 ° C. for 6 hours.

(Ii) After dissolution, centrifugation was performed at 3,000 x g for 10 minutes to obtain a supernatant to obtain a DMSO soluble material.

(Iii) The DMSO solubles obtained above were concentrated using a rotary vacuum concentrator.

(Iii) The concentrated sample was dried using a lyophilizer to obtain a DMSO extract.

2-3: Preparation of Distilled Water Extract, Methanol Extract and Acetone Extract

The effective material was extracted from the stem cells prepared in Example 1 step by step as follows.

(Iii) 500 mL of distilled water was added to 500 g of the cell line isolated in Example 1 from which the culture solution was removed, and dissolved by stirring at 50 ° C. for 6 hours.

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

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

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

(Iii) After obtaining the methanol soluble substance, 500 mL of acetone was added to the remaining methanol insoluble substance, followed by stirring at room temperature for 6 hours to dissolve it.

(Iii) After dissolution, the mixture was centrifuged at 3,000 x g for 10 minutes to obtain a supernatant to obtain an acetone soluble material.

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

(Iii) The concentrated sample was dried using a lyophilizer and dissolved in cell culture medium, methanol and acetone to obtain distilled water extract, methanol extract and acetone extract.

Cytotoxicity Check

To determine the range of concentration of use of the sample to be used for the test, cytotoxicity was checked as follows.

First, the human keratinocyte line (HaCaT: German Cancer Research Institute, Heidelberg, Germany) was cultured in DMEM medium (Welgene, Korea) to which 10% FBS was added. After inoculating 3 × 10 ⁴ / well (96-well plate) and incubating for 24 hours, the DMSO extract of the stem cell derived from the Ginkgo biloba forming layer obtained in Example 2 was prepared at a concentration of 1 ppm, 10 ppm and 100 ppm, and Stem cells were removed in Example 2, and the obtained culture was treated with FBS-free DMEM medium at concentrations of 0.1%, 1% and 10% (v / v) for 24 hours.

Survival rate of human keratinocytes was measured by absorbance of 450 nm after removing the test substance, washing with medium (PBS) and reacting for 1 to 2 hours in FMEM-free DMEM including 10% of WST-1 solution.

As a result, as shown in Figure 12, it was confirmed that the toxicity does not show a decrease in the survival rate of HaCaT cells at a concentration of less than 100 ppm. In Figure 12, Cell represents the DMSO extract of stem cells, and media represents the culture solution removed when the stem cell extract was prepared.

Collagenase Induced by UV Irradiation MMP -1) Confirmation of generation inhibitory effect

When MMP is increased by UV exposure, the increased MMPs break down collagen in the skin to form skin wrinkles, and the expression of MMP-1 increased by ultraviolet rays is inhibited by Ginkgo biloba stem-derived stem cell extract or its culture. The following experiment was conducted to confirm the effect.

First, fibroblasts (MCTT, Korea) were inoculated in 3 × 10 ⁴ / well (24-well plate) and incubated for 12 hours, followed by 6 hours starvation in FBS-free DMEM medium. Washed with DPBS buffer and irradiated with UVA (365 nm) 10 J / cm ² , each sample (DMSO extract of stem cells derived from cambium cambium: 10 ppm; culture medium: 10% (v / v)) was added to DMEM serum free. The medium was treated for 48 hours by concentration. The cultured medium was collected and the amount of MMP-1 was measured by western blot assay in the first test and ELISA assay in the second test. At this time, the positive control group used retinoic acid (RA) of 0.01 μM.

As a result, as shown in Figure 13, the stem cell extract and the culture medium according to the present invention appeared to inhibit the expression of MMP-1 more effectively than the retinoic acid known to have a strong wrinkle improvement effect appeared to have anti-wrinkle and improvement effect . In particular, both stem cell extracts and cultures were found to exhibit a lower MMP-1 expression inhibitory effect than when not treated with UV. In other words, the stem cells and the culture medium according to the present invention showed a superior inhibitory ability than retinoic acid was found to be very useful as an anti-aging cosmetic composition.

UV Of active oxygen inhibition effect by

In order to confirm the effect of increased free radicals induced by ultraviolet light is inhibited by stem cells derived from the cambium of cambium, the following experiment was performed.

First, the human keratinocyte line (HaCaT: German Cancer Research Institute, Heidelberg, Germany) was prepared in a ³ x 10 ⁴ in 96 black well plate. Dispense into cells / well and sample (2 and 10 ppm of DMSO extract of stem cells derived from the cambium cambium; 5% (v / v) of the culture solution of stem cells derived from the cambium cambium (37%, 5% CO). Incubated for 18 hours under conditions of _2. At this time, 10 mM of NAC (N-acetyl-cysteine) was treated as a positive control group, and after incubation, the medium was removed, and then 50 μM of 2'7'-dichlorodihydrofluorescin diluted with HBSS. Diacetate (DCFH-DA, Sigma) was added and incubated for 30 minutes, washed twice with HBSS, irradiated or irradiated with UVB at a dose of 50 mJ / cm ² , and incubated for 2 hours using a fluorescence microplate reader. The fluorescence intensity was measured at the excitation wavelength of 485 nm and the emission wavelength of 535 nm, and the rate of increase and decrease of the DCF fluorescence intensity was calculated in multiples of the control group (non-UV irradiated group).

As a result, as shown in Figure 14, in the case of the stem cell line extract according to the present invention, it was confirmed that the active oxygen inhibitory effect increased by UV stress. In particular, the group treated with 10 ppm showed much better inhibitory effect than NAC (N-Acetyl-L-Cysteine), a positive control group that is known to have active oxygen inhibitory effect by UV stress.

In addition, as shown in Figure 15, it was confirmed that the stem cell line culture medium according to the present invention also has an active oxygen inhibitory effect increased by UV stress. In particular, it can be seen that compared with the positive control group NAC (N-Acetyl-L-Cysteine), which is known to have an active oxygen inhibitory effect by UV stress, it showed that the active oxygen inhibitory ability was almost similar.

Determination of melanin formation inhibition effect

In order to confirm the melanin formation inhibitory effect of the stem cells derived from the cambium of the ginkgo biloba, the following experiment was performed.

First, Murine melanoma (B-16 F1, Korea Cell Line Bank, Korea) was inoculated with 1 × 10 ⁵ per well in a 12-well plate with RPMI1640 medium containing 10% FBS, followed by 5% CO ₂ at 37 ° C. Cells were incubated until about 80% adhered to the well bottom. The medium was removed, and each sample (DMSO extract of stem cell derived from the cambium cambium: 50 ppm; culture medium: 10% (v / v)) was replaced with a medium diluted to an appropriate concentration, followed by 5% CO ₂ , 37 ° C. It was incubated for a certain time under. At this time, Arbutin was treated with 10 mM as a positive control group, and the negative control group was a sample addition group. The cells from which the medium was removed were washed with PBS and treated with trypsin to recover the cells. The recovered cells were centrifuged at 5,000 to 10,000 rpm for 10 minutes, and then the supernatant was removed to obtain pellets. The pellets were dried at 60 ° C, and then 100 μl of 1 M NaOH containing 10% DMSO was added. Got my melanin This solution was measured by absorbance at 490 nm with a microplate reader (Tecan Infinite M200, Austria) to determine the amount of melanin per protein. Protein quantification was done using the Bradford method

As a result, as shown in Figure 16, when treated with the stem cell extract and the culture medium according to the present invention, it was measured to show a melanin forming activity as low as 60% compared to the control, thereby inhibiting melanin formation as a cosmetic composition for whitening It has been found to be useful.

Manufacturing example  One. Pharmaceutical Manufacturing example

Formulation example  1-1: Preparation of Tablet

100 mg of the stem cell extract prepared in Example 2 was mixed according to the conventional tablet preparation method by mixing cornstarch 100 mg, lactose 100 mg and magnesium stearate 2 mg.

Formulation example  1-2: Preparation of Capsules

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

Formulation example  1-3: Preparation of Syrup

100 mL of syrup was prepared according to a conventional method of preparing a liquid formulation with the content of 1 g of 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 Castro oil to prepare an injection containing a mixed extract at a concentration of 0.1%.

Manufacturing example  2. Preparation of Functional Foods

Manufacturing example  2-1: Preparation of Functional Beverage

After dissolving 200 mg of the stem cells obtained in Example 1 in 96 ml of water, 500 mg of vitamin C as an adjuvant, 1 g of citric acid and oligosaccharides as a copulating agent were added, and 0.05 g of sodium benzoate was added as a preservative. The amount was added to 100ml to prepare a functional beverage.

Manufacturing example  2-2: Preparation of Functional Beverage

After dissolving 200 mg of the stem cell extract prepared in Example 2 in 96 mL of water, 500 mg of vitamin C as an adjuvant, 1 g of citric acid and oligosaccharides as a copulating agent were added, and 0.05 g of sodium benzoate was added as a preservative. The amount was added to 100 mL to prepare a functional beverage.

Manufacturing example  3. Manufacturing of functional cosmetics

Manufacturing example  3-1: Preparation of Latex

6.2 mg of stem cell extract prepared in Example 2, 1,3-butylene glycol 6.5 mg, glycerin 1.2 mg, D-panthenol 0.2 mg, ethanol 3.0 mg, carbomer 0.1 mg, stearic acid 1.5 mg, polysorbate 60 0.7 mg, lipophilic glyceryl stearate 0.6 mg, sorbitan sesquioleate 0.3 mg, cetearyl alcohol 0.6 mg, squalane 3.5 mg, carlic / capric triglyceride 3 mg, dimethicone 0.4 mg, small amount of preservative , According to the composition of the combination perfume, purified water (to be added to 100 mg total) was prepared in the usual manner.

Manufacturing example  3-2: Preparation of Cream

Stem cell extract prepared in Example 2 5.0 mg, 1,3-butylene glycol 7.0 mg, glycerin 1.0 mg, D- panthenol 0.1 mg, magnesium aluminum silicate 0.4 mg, stearic acid 2.0 mg, polysorbate 60 1.5 mg , Lipophilic glyceryl stearate 2.0 mg, sorbitan sesquioleate 1.5 mg, mineral oil 4.0 mg, cetearyl alcohol 3.0 mg, squalane 3.8 mg, carlic / capric triglyceride 2.8 mg, dimethicone 0.4 mg, Cream was prepared according to the conventional method according to the composition of xanthan gum amount, triethanolamine amount, tocopheryl acetate amount, small amount of preservative, combination perfume amount, purified water (added to 100 mg total).

As described above in detail a specific part of the content of the present invention, for those skilled in the art, such a specific description is only a preferred embodiment, which is not limited by the scope of the present invention Will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (34)

An anti-aging composition derived from the cambium of the family Ginkgoaceae , containing any one or more of stem cells derived from the cambium of the ginkgo family, its extract, its lysate, and its culture, which are innate undifferentiated cells.
The anti-aging composition according to claim 1, wherein the cambium-derived stem cells have at least one of the following characteristics:
(a) contains a large number of single cells or a small size of cell aggregates in suspension culture compared to the dedifferentiated callus of Ginkgo biloba;
(b) exhibit morphological features with multiple vacuoles;
(c) has mitochondria with increased activity compared to dedifferentiated callus of Ginkgo biloba;
(d) can grow faster and grow longer than de-differentiated callus of Ginkgo biloba; And
(e) Has low sensitivity to shear stress in bioreactors compared to dedifferentiated callus from Ginkgo biloba.
The anti-aging composition according to claim 2, wherein the cambium-derived stem cells have at least two of the properties of (a) to (e).
The anti-aging composition according to claim 2, wherein the cambium-derived stem-derived stem cells have at least three of the properties of (a) to (e).
The composition for anti-aging according to claim 2, wherein the cambium-derived stem cell has at least four of the properties of (a) to (e).
The anti-aging composition according to claim 2, wherein the cambium-derived stem cells have the characteristics of (a) to (e).
The anti-aging composition according to claim 1, wherein the cambium-derived stem cells are separated by a separation method comprising the following steps:
(a) obtaining a cambium-containing tissue from a Ginkgo biloba plant;
(b) culturing the obtained cambium-containing tissue in a medium; And
(c) obtaining the cambium-derived stem cells by separating the cells from the cambium.
According to claim 1, wherein the culture is the stem cells 3 to 5% (g / L) of raw sugar or sugar; Or methyl jasmonate, fungus extract, bacterial extract, yeast extract, chitosan, glucomanan, glucan, phenylalanine, benzoic acid, salicylic acid ), Arachidonic acid, STS, mevalonalonate N-benzolyglycine, ABA, SNP, IPP, BHT, CCC, ethephon, hipuic acid, ceric ammonium nitrate, AgNO _3, vanadyl sulfate (vanadyl sulfate), p - benzo amino acid containing any one or more of the (p -aminobenzoic acid), bra Sino steroid (brassinosteroids), sodium alginate (sodium alginate) and sodium acetate (sodium acetate) Antioxidant or anti-aging composition, characterized in that obtained by further culturing in the medium.
The anti-aging 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.
8. The anti-aging composition according to any one of claims 1 to 7, wherein the Ginkgo biloba is genus Ginkgo .
The method according to any one of claims 1 to 7, wherein the ginkgo biloba isGinkgo bilobaThe anti-aging composition characterized in that).
An anti-aging or whitening cosmetic composition derived from the formation layer of ginkgo biloba and containing any one or more of the stem cells derived from the formation layer of ginkgo biloba, its extract, its lysate, and its culture, which are innate undifferentiated cells.
The anti-aging or whitening cosmetic composition according to claim 12, wherein the cambium-derived stem cells have at least one of the following characteristics:
(a) contains a large number of single cells or a small size of cell aggregates in suspension culture compared to the dedifferentiated callus of Ginkgo biloba;
(b) exhibit morphological features with multiple vacuoles;
(c) has mitochondria with increased activity compared to dedifferentiated callus of Ginkgo biloba;
(d) can grow faster and grow longer than de-differentiated callus of Ginkgo biloba; And
(e) Has low sensitivity to shear stress in bioreactors compared to dedifferentiated callus from Ginkgo biloba.
The anti-aging or whitening cosmetic composition according to claim 13, wherein the stem cells derived from the cambium-derived layer of Ginkgo biloba have at least two of the properties of (a) to (e).
The anti-aging or whitening cosmetic composition according to claim 13, wherein the cambium-derived stem cells have at least three of the properties of (a) to (e).
The anti-aging or whitening cosmetic composition according to claim 13, wherein the stem cell derived from the cambium of Ginkgo biloba has at least four of the properties of (a) to (e).
The anti-aging or whitening cosmetic composition according to claim 13, wherein the stem cell of the cambium-derived layer has the characteristics of (a) to (e).
The anti-aging or whitening cosmetic composition according to claim 12, wherein the stem cells derived from the cambium-derived layer of Ginkgo biloba are separated by a separation method comprising the following steps:
(a) obtaining a cambium-containing tissue from a Ginkgo biloba plant;
(b) culturing the obtained cambium-containing tissue in a medium; And
(c) obtaining the cambium-derived stem cells by separating the cells from the cambium.
The method of claim 12, wherein the culture of the stem cells 3 to 5% (g / L) raw sugar or sugar; Or methyl jasmonate, fungus extract, bacterial extract, yeast extract, chitosan, glucomanan, glucan, phenylalanine, benzoic acid, salicylic acid ), Arachidonic acid, STS, mevalonalonate N-benzolyglycine, ABA, SNP, IPP, BHT, CCC, ethephon, hipuic acid, ceric ammonium nitrate, AgNO _3, vanadyl sulfate (vanadyl sulfate), p- amino-benzo acid containing any one or more of the (p -aminobenzoic acid), bra Sino steroid (brassinosteroids), sodium alginate (sodium alginate) and sodium acetate (sodium acetate) Anti-aging or whitening cosmetic composition, characterized in that obtained by further culturing in the medium.
The anti-aging or whitening cosmetic composition 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.
The cosmetic composition for anti-aging or whitening according to any one of claims 12 to 18, wherein the Ginkgo biloba is Ginkgo biloba.
The cosmetic composition for anti-aging or whitening according to any one of claims 12 to 18, wherein the Ginkgo biloba is Ginkgo biloba.
The anti-aging or whitening cosmetic composition according to claim 12, wherein the composition inhibits the production of MMP-1.
A functional food having an anti-aging ability, containing any one or more of the stem cells derived from the ginkgo biloba, a extract thereof, a lysate, and the culture, which are derived from the ginkgo biloba form and are not dedifferentiated.
25. The functional food having anti-aging activity according to claim 24, wherein the cambium-derived stem cells have at least one of the following characteristics:
(a) contains a large number of single cells or a small size of cell aggregates in suspension culture compared to the dedifferentiated callus of Ginkgo biloba;
(b) exhibit morphological features with multiple vacuoles;
(c) has mitochondria with increased activity compared to dedifferentiated callus of Ginkgo biloba;
(d) can grow faster and grow longer than de-differentiated callus of Ginkgo biloba; And
(e) Has low sensitivity to shear stress in bioreactors compared to dedifferentiated callus from Ginkgo biloba.
The functional food having anti-aging ability according to claim 25, wherein the cambium-derived stem cells have at least two of the properties of (a) to (e).
26. The functional food having anti-aging ability according to claim 25, wherein the stem cell derived from the cambium of Ginkgoaceae has at least three of the properties of (a) to (e).
The functional food having anti-aging ability according to claim 25, wherein the cambium-derived stem cells have at least four of the properties of (a) to (e).
26. The functional food having anti-aging ability according to claim 25, wherein the stem cell derived from the cambium of Ginkgoaceae has the characteristics of (a) to (e).
25. The functional food having anti-aging activity according to claim 24, wherein the cambium-derived stem cells are separated by a separation method comprising the following steps:
(a) obtaining a cambium-containing tissue from a Ginkgo biloba plant;
(b) culturing the obtained cambium-containing tissue in a medium; And
(c) obtaining the cambium-derived stem cells by separating the cells from the cambium.
The method of claim 24, wherein the culture of the stem cells 3 to 5% (g / L) of raw sugar or sugar; Or methyl jasmonate, fungus extract, bacterial extract, yeast extract, chitosan, glucomanan, glucan, phenylalanine, benzoic acid, salicylic acid ), Arachidonic acid, STS, mevalonalonate N-benzolyglycine, ABA, SNP, IPP, BHT, CCC, ethephon, hipuic acid, ceric ammonium nitrate, AgNO _3, vanadyl sulfate (vanadyl sulfate), p - benzo amino acid containing any one or more of the (p -aminobenzoic acid), bra Sino steroid (brassinosteroids), sodium alginate (sodium alginate) and sodium acetate (sodium acetate) Functional food having an anti-aging ability, characterized in that obtained by further culturing in the medium.
The functional food having anti-aging ability according to claim 24, 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.
31. The functional food having an antiaging effect according to any one of claims 24 to 30, wherein the Ginkgo biloba is Ginkgo biloba.
31. The functional food having anti-aging ability according to any one of claims 24 to 30, wherein the Ginkgo biloba is Ginkgo biloba.

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