JP2013155122A - Stem cell activator - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new agent capable of effectively activating stem cells.SOLUTION: There is provided a stem cell activator containing niaouli oil as an active ingredient.

Description

  The present invention relates to an agent capable of effectively activating stem cells.

  A stem cell is a cell that has two properties: a multipotency that produces a cell differentiated into a plurality of cells, and a self-replicating ability that produces the same cell by cell division. Stem cells derived from embryos, which are the early stages of development of fertilized eggs, are called embryonic stem cells (ES cells). Although human ES cells are expected to be used for regenerative medicine, the creation of new human ES cells has not been approved due to the ethical problem of using fertilized eggs.

  In recent years, artificial pluripotent stem cells (iPS cells) have attracted attention as cells having properties similar to ES cells. However, the production of iPS cells has many problems from the viewpoints of cell canceration, production efficiency, and the like. On the other hand, somatic stem cells having the ability to differentiate into a specific tissue are obtained from a patient's own body tissue, for example, bone marrow, and therefore have no ethical problems like embryonic stem cells.

  It is well known that the skin has epidermal stem cells (Non-patent Document 1) in the basal layer of the epidermis, and hair follicle epithelial stem cells (Non-patent Document 2) and skin pigments exist in a region called a bulge region of the hair follicle. It has been reported that stem cells (Non-patent Document 3) exist. On the other hand, in the dermis, elongated spindle-shaped fibroblasts are present in the fiber component mainly composed of collagen, but it is not clear whether stem cells are present in the dermal fibroblasts. Moreover, although it is known that skin-derived precursor cells (SKP) that differentiate into a plurality of cell lines such as fat, glia, cartilage, and muscle exist in the dermis (Non-patent Document 4), the dermis The association between fibroblasts and SKP is not clear.

  Mesenchymal stem cells isolated from bone marrow as progenitor cells of fibroblasts (Non-patent Document 5) are used as various cells belonging to the mesenchymal system (bone cells, muscle cells, chondrocytes, tendon cells, fat cells, etc.). Because it differentiates, it is expected to be applied to regenerative medicine, such as reconstruction of bones, blood vessels, and muscles. Recently, the possibility of existing in many tissues having mesenchymal tissue has been clarified, and mesenchymal stem cells have also been isolated from fat, umbilical cord blood, placenta and the like (Non-Patent Documents 6 to 6). 8).

According to recent knowledge, mesenchymal stem cells are known to exist in blood vessels throughout the body as vascular pericytes (pericytes), and work to stabilize blood vessels and maintain tissue homeostasis (Non-patent Document 9). And 10).
Further, when a blood vessel is destroyed at or near a tissue damage site, mesenchymal stem cells that are vascular pericytes (pericytes) proliferate away from the blood vessel and supply lost cells (Non-Patent Document) 11-14), it releases biologically active factors to protect tissues (Non-Patent Documents 15 to 19), and acts on the repair and regeneration of damaged tissues. These secretory factors have the effect of strongly suppressing immunity in addition to the effects of angiogenesis and anti-apoptosis (Non-patent Documents 21 and 22), and suppress the destruction of damaged tissues via T cells and B cells ( Non-patent documents 9 and 22) have also been reported.
Furthermore, mesenchymal stem cells are also known to exhibit antifibrotic effects (Non-patent Documents 23 and 24) and effects on multiple sclerosis and diabetes (Non-patent Document 9).

  On the other hand, it is becoming clear that chronic inflammation is a basic pathological condition common to various diseases (for example, metabolic syndrome, arteriosclerotic disease, cancer, neurodegenerative disease, autoimmune disease, etc.) (Non-patent Document 25). . For example, it has been reported that endothelial inflammation and insulin resistance are induced by chronic inflammation and cause various diseases such as diabetes and arteriosclerotic diseases (Non-patent Document 26). Furthermore, it has also been clarified that obese adipose tissue itself changes to inflammatory (Non-patent Documents 27 to 29). Since chronic inflammation occurs around blood vessels, it is considered that disruption of the interaction between mesenchymal stem cells, which are vascular pericytes (pericytes), and blood vessels also occurs in chronic inflammation.

  Based on the above findings, if the production and stabilization of mesenchymal stem cells can be promoted, various types of blood vessel stabilization, tissue homeostasis, repair and regeneration of damaged tissues, antifibrosis, multiple sclerosis, diabetes, etc. It is considered to be extremely effective for various uses such as prevention / treatment of diseases and prevention / amelioration of various conditions based on chronic inflammation such as metabolic syndrome.

  Mesenchymal stem cells have been shown to be present in fat as well as bone marrow, umbilical cord blood, and placenta. In addition, the present inventors have clarified that mesenchymal stem cells exist in the dermis as well as subcutaneous fat (Patent Document 1: International Publication No. 2011/034106). Considering the action of the mesenchymal stem cells described above, it is considered that the mesenchymal stem cells are activated in the dermis and subcutaneous fat, and are effective for improving the skin condition, regeneration and the like.

International Publication No. 2011/034106

Watt FM, J Dermatol Sci, 28: 173-180, 2002 Cotsarelis G et al., Cell, 57: 201-209, 1989 Nishimura EK et al., Nature, 416: 854-860, 2002 Wong CE al., J Cell Biol, 175: 1005-1015, 2006 Pittenger MF et al., Science, 284: 143-147, 1999 Park KW et al., Cell Metab, 8: 454-457, 2008 Flynn A et al., Cytotherapy, 9: 717-726, 2007 Igura K et al., Cytotherapy, 6: 543-553, 2004 da Silva Meirelles L et al., Stem Cells, 2008 Sep; 26 (9): 2287-2299 da Silva Meirelles L et al., J Cell Sci, 2006; 119: 2204-2213 Dai WD et al., Circulation, 2005; 112: 214-223 Fazel S et al., J Thorac Cardiovasc Surg, 2005; 130: 1310-1318 Noiseux N et al., Mol Ther, 2006; 14: 840-850 Zhao LR et al., Exp Neurol, 2002; 174: 11-20 Gnecchi M et al., Nat Med, 2005; 11: 367-368 Kinnaird T et al., Circ Res, 2004; 94: 678-685 Kinnaird T et al., Circulation, 2004; 109: 1543-1549 Tang YL et al., Ann Thorac Surg, 2005; 80: 229-237 Zhang M et al., FASEB J, 2007; 21: 3197-3207 Le Blanc K et al., J Intern Med, 2007; 262: 509-525 Uccelli A et al., Trends Immunol, 2007; 28: 219-226 Caplan AI et al., J Cell Biochem, 2006; 98: 1076-1084 Fang BJ et al., Transplantation, 2004; 78: 83-88 Ortiz LA et al., Proc Natl Acad Sci USA, 2003; 100: 8407-841 Yoshihiro Ogawa, Experimental Medicine, 28: 1680-1687, 2010 Medzhitov R, Nature, 454: 428-35, 2008 Hotamisligil GS, Nature, 444 (7121): 860-7, 2006 Wellen KE et al., J Clin Invest, 115 (5): 1111-9, 2005 Tonami Takayoshi et al., Experimental Medicine, 28: 1717-1723, 2010

  This invention is made | formed in view of the said background, The subject is providing the agent which can activate a stem cell effectively.

  As a result of examining various materials and screening agents that can effectively activate stem cells, the present inventors have found that near uri oil exhibits a remarkable stem cell activation action, and to achieve the present invention. It came.

That is, the gist of the present invention includes the following.
[1] A stem cell activator containing near uri oil as an active ingredient.
[2] The stem cell activator according to [1], wherein the stem cells are mesenchymal stem cells.
[3] The stem cell activator of [1] or [2] used as an external preparation.
[4] A cosmetic containing the stem cell activator of [1] to [3].

  According to the present invention, an agent capable of effectively activating stem cells is provided.

Fig. 1 (a) is a graph showing the effect of addition of plant-derived oil to the medium on the respiratory activity of fat-derived stem cells, and Fig. 1 (b) shows the effect of the addition on the respiratory activity of fibroblasts. It is a graph which shows.

[Near uri oil]
“Near cucumber” refers to Melaleuca viridiflora, an evergreen tree in the family Myrtaceae. Note that “near cucumber” is sometimes referred to as “near cucumber”, but “near cucumber” in this specification includes “near cucumber”. Near-uri oil is oil extracted from the whole plant or part of near-uri.

  There are examples of near uri oil that have been proposed to enhance versican expression / hair growth (JP 2005-200383, etc.), estrogen-like action (JP 2006-241044, etc.). In particular, it has not been known so far to have an activation effect on stem cells, and such an effect has been found for the first time by the inventors.

[Oil extraction method]
Near uri oil (hereinafter may be collectively referred to as “plant-derived oil” or simply “oil”) is not limited, but can be prepared by the following method, for example.

  The plant-derived oil material is a plant containing near cucumber. The whole or part of the plant may be used. In the case of a plant part, one or more types selected from flowers, flower spikes, fruit spikes, pericarps, fruits, stems, leaves, branches, branches and leaves, trunks, bark, rhizomes, root barks, roots, seeds and the like can be mentioned. These materials may be subjected to extraction as they are, or may be extracted after being subjected to processing such as pressing, drying, and pulverization.

  Plant-derived oil is extracted from the above materials using a solvent. Examples of the solvent include water and organic solvents. Examples of the organic solvent include lower (for example, C1-6) alcohols, polyol solvents, and the like. Examples of the lower alcohol include methanol, ethanol, propanol and the like. Examples of the polyol solvent include ethylene glycol and propylene glycol. These solvents may be used alone or in a combination of two or more in any combination and ratio.

  Although the method for extracting oil (oil component) from the above materials is not limited, typical methods include solvent extraction and steam distillation. In the solvent extraction, various fats and oils components that can be dissolved in a solvent (particularly lower alcohol, preferably ethanol) are extracted, but in the steam distillation, volatile fats and oils components are mainly extracted. Although any technique is used in the present invention, a plant-derived oil extracted by steam distillation is preferred.

  The steam distillation is distillation using water as a solvent. However, the above-mentioned other solvents may be appropriately mixed and used as long as the distillation is not hindered. Although the material may be distilled as it is, it may be subjected to distillation after the above-described direct extraction, filtration or the like. In addition, you may add processing, such as filtration, concentration, and distillation by a conventional method.

[Stem cell activator]
The stem cell activator of the present invention (hereinafter sometimes referred to as “the agent of the present invention”) contains near uri oil (plant-derived oil) as an active ingredient. When two or more active ingredients are contained, the combination and ratio thereof are arbitrary.

  Stem cells activated by the stem cell activator of the present invention are not limited, but mesenchymal stem cells (MSCs) are preferable. Examples of mesenchymal stem cells include adipose-derived stem cells (ASC or ADSC), dermis-derived stem cells, and the like. As mentioned above, the activation of mesenchymal stem cells can stabilize blood vessels, maintain tissue homeostasis, repair and regenerate damaged tissues (especially dermal regeneration), anti-fibrosis, multiple sclerosis, diabetes, etc. It is known to be extremely effective for uses such as prevention and treatment of various diseases, prevention and improvement of various conditions based on chronic inflammation such as metabolic syndrome. Therefore, the agent of the present invention can be used very effectively for these applications.

“Activation” of a stem cell in the present invention refers to an action of promoting and / or activating stem cells. The method for verifying the activation effect of the stem cell is not limited. For example, a method for measuring the respiratory activity of the stem cell using Alamar blue ^{(registered trademark)} (Invitrogen) or the like employed in Examples described later, MTT (3- (4, 5-di-methylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, yellow tetrazole) test and the like.

  The agent of the present invention may be composed only of the above-mentioned plant-derived oil which is an active ingredient, but the above-mentioned plant-derived oil is used as one or more other components such as excipients, carriers and / or It can also be set as the composition combined with the diluent etc. The composition and form of the composition are arbitrary, and may be appropriately selected according to conditions such as active ingredients and uses. The said composition can be manufactured using a conventional method by the prescription combined with the excipient | filler, the support | carrier and / or diluent etc., and other components suitably according to the dosage form.

  Although the use of the agent of the present invention is not limited, for example, it can be applied to humans or animals as a cosmetic, quasi-drug or pharmaceutical for transdermal administration in the form of a skin external preparation. The dosage form of the external preparation for skin may be any form as long as it can be applied to the skin, but it is a liquid agent (solution system, solubilization system, suspension system / emulsion system, powder dispersion system, water-oil two-layer system, water- Oil-powder three-layer system, etc.), ointments, patches, lotions, gels, aerosols and the like.

  When the agent of the present invention is used as an external preparation for skin, the agent of the present invention may be mixed as it is or with other components according to the application as necessary. As other components, components usually used in pharmaceutical preparations and quasi drugs, such as excipients, binders, disintegrants, thickeners, dispersants, reabsorption accelerators, corrigents, buffers, surfactants Agent, solubilizer, preservative, emulsifier, tonicity agent, stabilizer, pH adjuster, antioxidant, UV protection agent, coloring material, aqueous component, various skin nutrients, sequestering agent, oil component, Alcohols, sugars, powder components, water and the like can be mentioned. Any of these components may be used alone, or two or more thereof may be used in any combination and composition.

  The agent of the present invention is preferably used for cosmetic applications such as cosmetics. When the agent of the present invention is used in cosmetics as an external preparation for skin, the form thereof is arbitrary. For example, lotion, emulsion, foundation, lipstick, lip balm, cleansing cream, massage cream, pack, hand cream, hand powder, body Examples include shampoos, body lotions, body creams, and bath cosmetics.

  When the agent of the present invention is used as a skin external preparation in cosmetics, pharmaceutical preparations, quasi-drugs, etc., the amount of the agent of the present invention is appropriately determined according to the type, purpose, form, usage method, etc. of the skin external preparation. I can decide. For example, the plant-derived oil as the active ingredient is preferably 0.00001 to 50% by weight, more preferably 0.0001 to 5% by weight, based on the entire external preparation for skin.

  In addition, the agent of the present invention may be blended in various foods and drinks and feeds (pet food etc.) and ingested by humans and animals.

  Specifically, when the PDGF-BB production enhancer of the present invention is blended in foods and drinks, feeds, etc., the blending amount (dry mass) of the plant body or the extract thereof is the type, purpose, form, usage method, etc. It can be determined appropriately according to For example, it is preferable to blend so that the plant-derived oil, which is an active ingredient per day for adults, is 0.00001 to 50% by weight, especially 0.001 to 10% by weight. In particular, when used as a food and drink for specified health use, the plant-derived oil as an active ingredient per day for adults is 0.001 to 10% by weight so that the predetermined effect of the active ingredient of the present invention is sufficiently exerted. It is preferable to blend so as to be.

  The food and drink and the feed can be in any form, and can be formed into, for example, granules, granules, pastes, gels, solids, or liquids. These forms include various known substances that are recognized to be contained in foods and drinks, such as binders, disintegrants, thickeners, dispersants, reabsorption accelerators, corrigents, buffers, and interfaces. Excipients such as activators, solubilizers, preservatives, emulsifiers, tonicity agents, stabilizers and pH adjusters can be included as appropriate.

  Formulation examples when the agent of the present invention is used for various applications are shown below. However, the present invention is not limited by the following formulation examples.

(Formulation example 1: lotion)
(Compounding ingredients) (mass%)
Water residue Ethanol 10
Butylene glycol 7
Dipropylene glycol 5
Glycerin 2
Polyethylene glycol (PEG-6) 0.5
Polyethylene glycol (PEG-32) 0.5
Hydroxyethyl cellulose 0.1
Stem cell activator (near uri oil) 0.2
Arginine 0.01
Izayoi Rose Extract 0.2
Hydrolyzed yeast extract 0.02
Sodium hyaluronate 0.01
Ichiyakuso Extract 0.2
Water-soluble collagen 0.2
Asenyaku extract 0.1
Polypropylene glycol-13decyltetradeceth-24 0.5
Sodium citrate 0.05
Sodium metaphosphate 0.05
Citric acid 0.05
Preservative appropriate amount Fragrance proper amount

(Formulation example 2: essence)
(Compounding ingredients) (mass%)
(Phase A)
Water residue Glycerin 6
Ethanol 5
Dipropylene glycol 5
Polyethylene glycol / polypropylene glycol-14
/ 7 dimethyl ether 3
Polyethylene glycol-400 1
Sodium polyacrylate 0.1
Xanthan gum 0.05
Stem cell activator (near uri oil) 0.3
Arginine 0.01
Keihi extract 0.1
Pearl protein extract 0.2
Sodium hyaluronate 0.01
Water-soluble collagen 0.2
Asenyaku extract 0.1
Novara extract 0.2
Carboxyvinyl polymer 0.3
Butylene glycol 3
Potassium hydroxide 0.15
(Acrylic acid / alkyl acrylate (C10-30) copolymer 0.1
Sodium metaphosphate 0.03
(Phase B)
Cyclomethicone 2.5
Dimethicone 2
Polyethylene glycol-60 hydrogenated castor oil 0.5
Preservative appropriate amount perfume appropriate amount (production method)
The homogeneously dissolved A phase and B phase were mixed at room temperature and homomixed.

(Formulation formulation example 3: emulsion)
(Compounding ingredients) (mass%)
(Phase A)
Water residue Butylene glycol 8
Ethanol 5
Dipropylene glycol 5
Glycerin 5
Dimethicone 2
2-Amino-2-methyl-1,3-propanediol (AMPD) 0.08
Carboxyvinyl polymer 0.15
EDTA-3Na 0.05
Xanthan gum 0.1
Stem cell activator (near uri oil) 0.1
Arginine 0.01
Yuzu Seed Extract 0.2
Hydrolyzed yeast extract 0.2
Sodium hyaluronate 0.05
Ichiyakuso Extract 0.3
Water-soluble collagen 0.2
(Phase B)
Behenyl alcohol 0.4
Pentaerythrityl tetraethylhexanoate 5
Polyethylene glycol-60 hydrogenated castor oil 0.5
Hydrogenated polydecene 2
Hydrogenated palm oil 0.5
Batyl alcohol 0.4
Palm kernel oil 0.4
Palm oil 0.1
Preservative appropriate amount perfume appropriate amount (Manufacturing method)
A phase (oil phase part) and B phase (aqueous phase part) were each heated to 70 ° C. and completely dissolved. After adding A phase to B phase and stirring well, it emulsified with the emulsifier. The obtained emulsion was cooled using a heat exchanger.

(Formulation example 4: emulsion)
(Compounding ingredients) (mass%)
(Phase A)
Water residue Glycerin 8
Ethanol 5
Dipropylene glycol 4
Hydrogenated polydecene 4
Polyethylene glycol-20 3
Carboxyvinyl polymer 0.09
Xanthan gum 0.05
Sodium metaphosphate 0.02
2-Amino-2-methyl-1,3-propanediol (AMPD) 0.03
Stem cell activator (near uri oil) 0.2
Arginine 0.01
Rose extract 0.3
Okra extract 0.2
Sodium hyaluronate 0.01
Water-soluble collagen 0.5
(Phase B)
Pentaerythrityl tetraethylhexanoate 3
Cetyl ethylhexanoate 2
Hydrogenated palm oil 2
Vaseline 1.5
Dimethicone 1
Polyethylene glycol stearate-5 glyceryl 1
Polyethylene glycol isostearate-60 glyceryl 1
Behenyl alcohol 0.5
Glyceryl diisostearate 0.4
Batyl alcohol 0.2
Preservative appropriate amount perfume appropriate amount (Manufacturing method)
A phase (oil phase part) and B phase (aqueous phase part) were each heated to 70 ° C. and completely dissolved. After adding A phase to B phase and stirring well, it emulsified with the emulsifier. The obtained emulsion was cooled using a heat exchanger.

(Formulation Example 5: Ointment)
(Compounding ingredients) (mass%)
Stem cell activator (near uri oil) 2.0
Stearyl alcohol 18.0
Owl 20.0
Polyoxyethylene (20) monooleate 0.25
Glycerin monostearate 0.3
Vaseline 40.0
Purified water residue (Production method)
Stem cell activator (near uri oil) is added to purified water and dissolved, and kept at 70 ° C. (aqueous phase). The remaining components are mixed and dissolved at 70 ° C. (oil phase). An oil phase was added to the aqueous phase, and the mixture was uniformly emulsified with a homomixer and cooled to obtain an ointment.

(Formulation example 6: patch)
(Compounding ingredients) (mass%)
Stem cell activator (near uri oil) 1.0
Ester oil ("Panasate 875R") 2.5
Squalane 1.0
dl-camphor 0.07
Polyoxyethylene (60 mol) hydrogenated castor oil 1.2
Concentrated glycerin 5.0
Gelatin 1.2
Polyvinylpyrrolidone 0.6
Methyl paraben appropriate amount d-sorbitol solution 35.0
Aluminum hydroxide 0.2
Sodium sulfite appropriate amount Sodium edetate appropriate amount Citric acid appropriate amount Carboxyvinyl polymer 0.22
Sodium polyacrylate 0.24
Sodium carboxymethylcellulose 2.8
Kaolin 1.0
Purified water remaining

(Formulation Formulation Example 7: Drink)
(Compounding ingredients) (in mg / 50 mL)
Konagi hot water extract (dry mass) 10
Collagen peptide 5.0
Pearl barley extract (dry mass) 2.0
Reduced maltose starch syrup 28
Erythritol 8.0
Citric acid 2.0
Stem cell activator (near uri oil) 1.0
N-acetylglucosamine 1.0
Hyaluronic acid 0.5
Vitamin E 0.3
α-Lipoic acid 0.2
Coenzyme Q10 1.2
L-proline 2.0
Ceramide (konjac extract) 0.4
Water remaining

  Next, the present invention will be described in more detail with reference to examples. In addition, this invention is not limited at all by the following examples.

[material]
As the near-uri oil, Biolandes Niaouli Melaleuca Oil, which is an extract (essential oil) obtained by steam distillation of near-uri (Melaleuca viridiflora), was used.
After dissolving near-uri oil in ethanol, a medium (oil-added medium) was prepared by adding the obtained ethanol solution to MesenPRO (+) medium. The near-uri oil concentration in the medium was adjusted to 0.005% by weight in the medium. The ethanol concentration relative to the medium was 0.1% by weight.
As a control, a medium (oil non-added medium) in which no oil was added and ethanol alone was added at a concentration of 0.1% by weight in the medium was prepared.

[Cell culture]
In 24-well plates, human adipose-derived stem cells (mesenchymal MesenPRO for stem cell culture (+) medium (Invitrogen) passage 5 generations at), to be about 2 × 10 ⁴ cells per well MesenPRO (+ ) Seeding in medium. After 24 hours, the medium in each well was replaced with the above oil-added medium or oil-free medium, and further cultured for 24 hours.
Moreover, it culture | cultivated similarly using a human fibroblast instead of a human fat origin stem cell.

[Respiratory activity measurement]
As an indicator of whether the cultured cells were activated, the respiratory activity of the cells was measured using Alamar blue ^{(registered trademark)} (Invitrogen). Alamar blue ^{(registered trademark)} is a pigment that serves as an indicator of the respiratory activity of cells, and is blue (non-fluorescent) in the oxidized form, but changes to red (fluorescent) when reduced by the electron transport system. Therefore, when added to a cell culture medium, it becomes a reduced form when the cell has a high respiratory activity and an oxidized form when the cell has a low respiratory activity. Therefore, the respiratory activity of the cells can be examined by measuring red color (fluorescence).
The respiratory activity was measured according to the Alamar blue manual. Specifically, after culturing the cells for 24 hours in the presence of an oil-added medium or an oil-free medium, the medium in each well was replaced with an Alamar blue dye-containing MesenPRO (+) medium and incubated for 3 hours. The fluorescence intensity (excitation wavelength 545 nm, detection wavelength 590 nm) was measured. Each test was performed in triplicate (n = 3), and the measurement of respiratory activity obtained in the presence of each oil was subjected to Student's t-test against the control (no oil added) to verify its significance. .

[result]
The results are shown in the graph of FIG. FIG. 1 is a graph showing the effect of adding plant-derived oil to a medium on the respiratory activity of fat-derived stem cells.
As is clear from FIG. 1, near urioil oil was found to have a significant activation effect on the respiratory activity of adipose-derived stem cells compared to the control (oil-free medium) at a concentration of 0.005 wt% in the medium.

  The present invention has significant applicability in various fields such as pharmaceuticals, cosmetics, and foods that require activation of stem cells.

Claims (4)

  A stem cell activator containing near uri oil as an active ingredient.   The stem cell activator according to claim 1, wherein the stem cells are mesenchymal stem cells.   The stem cell activator according to claim 1 or 2, which is used as an external preparation.   Cosmetics containing the stem cell activator according to any one of claims 1 to 3.

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