JP2017155024A - Cell growth promoter - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel agent for promoting novel fibroblast proliferation.SOLUTION: According to the present invention, a cell growth promoter comprises a cellulose fiber as an active ingredient, and the cellulose fiber has a number average fiber diameter of 2 to 150 nm. The cellulose fiber has a cellulose type I crystal structure, and a hydroxyl group at position C6 of each glucose unit in the molecule of cellulose is modified to a carboxyl group by selective oxidation, resulting in a carboxyl group ratio of 0.6 to 2.5 mmol/g.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a novel cell growth promoter.

In recent years, various fine celluloses obtained by physically finely processing cellulose itself have been developed. For example, among them, cellulose microfibrils are ultrafine fibers having a nano-level width, have high crystallinity, and have excellent characteristics that cannot be obtained artificially.
However, cellulose microfibrils cannot be nano-dispersed in an aqueous solvent because the microfibrils are strongly hydrogen bonded to each other.

Therefore, cellulose nanofibers that have been oxidized using a cooxidant in the presence of an N-oxyl compound have been developed. In the cellulose nanofiber, the surface of the microfibril is ionized and easily nano-dispersed in water by electrostatic repulsion.
This cellulose nanofiber has a characteristic property that it is uniformly dispersed without dissolving in water, and becomes a gel having a thixotropic property that becomes a high-viscosity gel when standing and becomes liquid when flowing. Various applications such as cosmetics in general (for example, Patent Document 1) and color materials such as paints and inks (for example, Patent Document 2) have been proposed in order to maintain the effect after spraying in a gel form. Yes.

Japanese Patent No. 5269512 Japanese Patent No. 5545775

  The cellulose nanofiber having such characteristic properties is expected to have further functionality.

The present invention has been made in view of the above problems, and an object of the present invention is to provide a novel fibroblast proliferation promoter.
Another object of the present invention is to provide a fibroblast growth promoter as a novel use of cellulose nanofibers.

In order to find out further added value of the above-mentioned cellulose nanofibers, the present inventors have searched for the functionality thereof. As a result, the present inventors have shown that they have a growth promoting effect on fibroblasts and an effect of promoting fibroblast migration. It came to the headline and this invention.
In order to achieve the above object, the cell growth promoter of the present invention comprises cellulose fiber as an active ingredient, and the cellulose fiber has a number average fiber diameter of 2 to 150 nm, and the cellulose has a cellulose I-type crystal structure. And the hydroxyl group at the C6 position of each glucose unit in the cellulose molecule is selectively oxidized and modified to a carboxyl group, whereby the carboxyl group has a ratio of 0.6 to 2.5 mmol / g. Take the configuration.

The cellulose fiber may be oxidized using a co-oxidant in the presence of an N-oxyl compound.
The cell growth promoter is a dispersion in which the cellulose fibers are dispersed in an aqueous solvent, and the concentration of the cellulose fibers in the cell growth promoter is 6.0 × 10 ⁻⁵ wt% to 5.0%. It is good to be weight%.
The cell growth promoter may be a fibroblast growth promoter.

  According to the present invention, the number average fiber diameter is 2 to 150 nm, the cellulose has a cellulose I-type crystal structure, and the hydroxyl group at the C6 position of each glucose unit in the cellulose molecule is selectively oxidized. To provide a cell growth promoter excellent in cell growth promotion effect and cell migration promotion effect possessed by a cellulose fiber modified with a carboxyl group and thereby having a carboxyl group ratio of 0.6 to 2.5 mmol / g Can do.

In the cell growth promotion effect confirmation test of Experiment 1, it is a graph which shows the time-dependent change of the cell proliferation after adding the cellulose fiber which concerns on one Example of this invention. In the cell growth promotion effect confirmation test of Experiment 1, it is a graph which shows the increase rate of the cell number 3 days after the cellulose fiber addition which concerns on one Example of this invention. In the cell migration test of Experiment 2, it is a photograph which shows the external appearance of the wound before and behind culture | cultivation after scratching. In the cell migration test of Experiment 2, it is a graph which shows the change rate which shows the ratio of the area where the wound closed after culture | cultivation.

Hereinafter, the cell growth promoter according to an embodiment of the present invention will be described.
The cell growth promoter according to the present invention contains a specific cellulose fiber as an active ingredient.
The specific cellulose fiber is a cellulose fiber having a number average fiber diameter of 2 to 150 nm. The cellulose has a cellulose I-type crystal structure, and the hydroxyl group at the C6 position of the glucose unit in the cellulose molecule is selectively used. It is a fine cellulose fiber oxidized and modified to carboxyl group. This means that the cellulose fiber is a fiber obtained by subjecting a naturally-derived cellulose solid raw material having an I-type crystal structure to surface oxidation and refinement. That is, in the process of biosynthesis of natural cellulose, nanofibers called microfibrils are first formed almost without exception, and these form multiple bundles to form a higher-order solid structure. In order to weaken the hydrogen bond between the surfaces that are the driving force of the water, a part of the hydroxyl group (the hydroxyl group at the C6 position of each glucose unit in the cellulose molecule) is oxidized and converted into a carboxyl group.

  The cellulose constituting the specific cellulose fiber has an I-type crystal structure, for example, in the diffraction profile obtained by wide-angle X-ray diffraction image measurement, in the vicinity of 2 theta = 14-17 ° and 2 theta = 22-23. It can be identified from the fact that there are typical peaks at two positions near °.

  The specific cellulose fiber has a number average fiber diameter of 2 to 150 nm. The number average fiber diameter is preferably 2 to 100 nm, particularly preferably 3 to 80 nm. If the number average fiber diameter is less than the above range, it will essentially dissolve in the dispersion medium, and conversely if the number average fiber diameter exceeds the above range, the cellulose fibers will settle and contain the cellulose fibers. This is because the functionality cannot be expressed.

  The number average fiber diameter of the specific cellulose fiber can be measured, for example, as follows. That is, an aqueous dispersion of fine cellulose having a solid content of 0.05 to 0.1% by weight was prepared, and the dispersion was cast on a carbon film-coated grid that had been subjected to a hydrophilization treatment. (TEM) observation sample. In addition, when the fiber of a big fiber diameter is included, you may observe the scanning electron microscope (SEM) image of the surface cast on glass. Then, observation with an electron microscope image is performed at a magnification of 5000 times, 10000 times, or 50000 times depending on the size of the constituent fibers. At that time, an axis having an arbitrary vertical and horizontal image width is assumed in the obtained image, and the sample and observation conditions (magnification, etc.) are adjusted so that 20 or more fibers intersect the axis. Then, after obtaining an observation image that satisfies this condition, two random axes, vertical and horizontal, per image are drawn on this image, and the fiber diameter of the fiber that intersects the axis is visually read. In this way, images of at least three non-overlapping surface portions are taken with an electron microscope, and the fiber diameter values of the fibers intersecting with each of the two axes are read (thus, at least 20 × 2 × 3 = 120). Information on the fiber diameter of the book is obtained). The number average fiber diameter is calculated from the fiber diameter data thus obtained.

  The specific cellulose fiber has a hydroxyl group at the C6 position of each glucose unit in the cellulose molecule selectively oxidized and modified to a carboxyl group, whereby the carboxyl group ratio is 0.6 to 2.5 mmol. / G. The content of the carboxyl group is preferably in the range of 1.0 to 2.0 mmol / g from the viewpoint of shape retention performance and dispersion stability. In addition, when the amount of the carboxyl group is less than the above range, the dispersion stability of the cellulose fiber is poor and precipitation may occur, and conversely, when the amount of the carboxyl group exceeds the above range, the water solubility becomes strong and sticky. A tendency to give a feeling of use comes to be seen.

  The measurement of the carboxyl group amount of the specific cellulose fiber is, for example, preparing 60 ml of a 0.5 to 1% by weight slurry from a cellulose sample precisely weighed on the dry weight, and adjusting the pH to about 2.5 with a 0.1 M hydrochloric acid aqueous solution. Then, 0.05M sodium hydroxide aqueous solution is dropped, and the electrical conductivity is measured. The measurement is continued until the pH is about 11. The amount of carboxyl groups can be determined from the amount of sodium hydroxide consumed in the neutralization step of a weak acid with a slow change in electrical conductivity (V) according to the following formula.

  Amount of carboxyl groups (mmol / g) = V (ml) × [0.05 / cellulose weight] (formula)

  In addition, adjustment of a carboxyl group amount can be performed by controlling the addition amount and reaction time of the co-oxidant used at the oxidation process of a cellulose fiber so that it may mention later.

  In the specific cellulose fiber, only the hydroxyl group at the C6 position of each glucose unit in the cellulose molecule on the fiber surface is selectively oxidized to a carboxyl group. Whether or not only the hydroxyl group at the C6 position of the glucose unit on the cellulose fiber surface is selectively oxidized to a carboxyl group can be confirmed by, for example, a 13C-NMR chart. That is, the 62 ppm peak corresponding to the C6 position of the primary hydroxyl group of the glucose unit, which can be confirmed on the 13C-NMR chart of cellulose before oxidation, disappears after the oxidation reaction, and instead, a peak derived from the carboxyl group at 178 ppm. Appears. In this way, it can be confirmed that only the C6 hydroxyl group of the glucose unit is oxidized to a carboxyl group.

  In the present invention, the specific cellulose fiber is particularly oxidized by using a co-oxidant in the presence of an N-oxyl compound such as 2,2,6,6-tetramethylpiperidine (TEMPO). The specific cellulose fiber can be easily obtained.

The cell to which the cell growth promoting agent according to the present invention is applied is not particularly limited, and the ability to proliferate eukaryotic cells of various species can be improved.
In particular, cells of humans or non-human animals (particularly mammals) are preferred as application targets.
In addition, somatic cells (skin fibroblasts, nerve cells, vascular endothelial cells, etc.) and germ cells are preferred as proliferation targets.
Stem cells may be targeted for medical value. Examples of such stem cells include embryonic stem cells, induced pluripotent stem cells (iPS cells), mesenchymal stem cells, neural stem cells, bone marrow stem cells, hematopoietic stem cells, and the like. From the viewpoint of cell proliferation, it is preferable to use stem cells that are in an undifferentiated state (that is, stem cells that have not been subjected to differentiation induction treatment).

In one embodiment, the cell growth promoter comprising the specific cellulose fiber of the present invention as an active ingredient may be a skin fibroblast proliferation promoter that promotes the proliferation of skin fibroblasts.
Fibroblasts are cells that are dispersed in almost all tissues of an animal individual and are components of fibrous connective tissue that play an important role in organ morphogenesis. That is, it is the most important cell for maintaining the function of the skin, and has no outstanding function in normal tissues.
When the tissue is damaged, fibroblasts migrate to the damaged part and start producing extracellular matrix such as collagen. It renews the extracellular matrix and plays an important role in the wound healing process such as cell-extracellular matrix interaction and wound contraction.
In general, in the healing process (wound healing) that occurs after an animal is injured, the proliferation of fibroblasts plays an important role in the repair of wounds and the like.
In the present invention, in Test Example 1 to be described later, since the specific cellulose fiber showed the dermal fibroblast proliferation promoting action, the specific cellulose fiber has the effect of accelerating the wound healing process and speeding up the healing of the trauma. It was confirmed.

That is, there are the following three stages in the process of healing skin damage (scratches).
That is, as a first step, blood stops due to platelet aggregation and vasoconstriction, and then macrophages (phagocytic cells) capture and clean the dead tissue on the wound surface. In the second step, repair by a granulation tissue mainly composed of collagen secreted by fibroblasts is started. In the third step, the granulation tissue changes to scar tissue, resulting in a stable wound.
The fibroblast proliferation promoter according to the present invention plays a role of increasing collagen by promoting fibroblast proliferation in the second step.

  Next, the production of the specific cellulose fiber will be described in more detail. The cellulose fiber is subjected to, for example, (1) an oxidation reaction step, (2) a purification step, (3) a dispersion step (a refinement treatment step), etc. Can be obtained. Hereinafter, each step will be described in order, and finally, (4) addition of other additives will be described.

(1) Oxidation reaction step After natural cellulose and an N-oxyl compound are dispersed in water (dispersion medium), a cooxidant is added to start the reaction. During the reaction, a 0.5 M aqueous sodium hydroxide solution is added dropwise to maintain the pH at 10 to 11, and the reaction is regarded as completed when no change in pH is observed. Here, the co-oxidant is not a substance that directly oxidizes a cellulose hydroxyl group but a substance that oxidizes an N-oxyl compound used as an oxidation catalyst.

  The natural cellulose means purified cellulose isolated from a cellulose biosynthetic system such as a plant, animal, or bacteria-producing gel. More specifically, softwood pulp, hardwood pulp, cotton pulp such as cotton linter and cotton lint, non-wood pulp such as straw pulp and bagasse pulp, bacterial cellulose (BC), cellulose isolated from sea squirt, seaweed Cellulose isolated from can be mentioned. These may be used alone or in combination of two or more. Among these, soft wood pulp, hardwood pulp, cotton pulp such as cotton linter and cotton lint, and non-wood pulp such as straw pulp and bagasse pulp are preferable. The natural cellulose is preferably subjected to a treatment for increasing the surface area such as beating, because the reaction efficiency can be increased and the productivity can be increased. In addition, if the natural cellulose that has been stored after being isolated and purified and not dried (never dry) is used, the microfibril bundles are likely to swell. This is preferable because the number average fiber diameter after the crystallization treatment can be reduced.

  The dispersion medium of natural cellulose in the above reaction is water, and the concentration of natural cellulose in the reaction aqueous solution is arbitrary as long as the reagent (natural cellulose) can be sufficiently diffused. Usually, it is about 5% or less with respect to the weight of the reaction aqueous solution, but the reaction concentration can be increased by using an apparatus having a strong mechanical stirring force.

  Examples of the N-oxyl compound include compounds having a nitroxy radical generally used as an oxidation catalyst. The N-oxyl compound is preferably a water-soluble compound, more preferably a piperidine nitroxyoxy radical, particularly 2,2,6,6-tetramethylpiperidinooxy radical (TEMPO) or 4-acetamido-TEMPO. preferable. The N-oxyl compound is added in a catalytic amount, preferably 0.1 to 4 mmol / l, more preferably 0.2 to 2 mmol / l.

  Examples of the co-oxidant include hypohalous acid or a salt thereof, hypohalous acid or a salt thereof, perhalogen acid or a salt thereof, hydrogen peroxide, a perorganic acid, and the like. These may be used alone or in combination of two or more. Of these, alkali metal hypohalites such as sodium hypochlorite and sodium hypobromite are preferable. And when using the said sodium hypochlorite, it is preferable in terms of reaction rate to advance reaction in presence of alkali bromide metals, such as sodium bromide. The addition amount of the alkali metal bromide is about 1 to 40 times mol, preferably about 10 to 20 times mol for the N-oxyl compound.

  The pH of the aqueous reaction solution is preferably maintained in the range of about 8-11. The temperature of the aqueous solution is arbitrary at about 4 to 40 ° C., but the reaction can be performed at room temperature (25 ° C.), and the temperature is not particularly required to be controlled.

  In order to obtain the target amount of carboxyl groups, the degree of oxidation is controlled by the amount of co-oxidant added and the reaction time. Usually, the reaction time is completed within about 5 to 120 minutes, at most 240 minutes.

  And after completion | finish of the said reaction, hydrochloric acid is added and it adjusts to neutrality (pH 6.0-8.0). For the purpose of improving long-term storage stability, reduction treatment may be performed with sodium borohydride or the like after the completion of the reaction.

(2) Purification step Next, purification is appropriately performed for the purpose of removing unreacted co-oxidant (such as hypochlorous acid) and various by-products. At this stage, the reactant fibers are usually not dispersed evenly to the nanofiber unit. Therefore, by repeating the usual purification method, that is, washing with water and filtration, the reactant fibers are highly purified (99% by weight or more). Use water dispersion.

  As the purification method in the purification step, any device may be used as long as it can achieve the above-described object, such as a method using centrifugal dehydration (for example, a continuous decanter). The aqueous dispersion of the reactant fibers thus obtained is in the range of approximately 10 wt% to 50 wt% as the solid content (cellulose) concentration in the squeezed state. Considering the subsequent dispersion step, if the solid content concentration is higher than 50% by weight, it is not preferable because extremely high energy is required for dispersion.

(3) Dispersion process (miniaturization process)
The reaction fiber (water dispersion) impregnated with water obtained in the purification step is dispersed in a dispersion medium such as water to perform a dispersion treatment. With the treatment, the viscosity increases, and a dispersion of finely pulverized cellulose fibers can be obtained. Then, a specific cellulose fiber can be obtained by drying the dispersion of the cellulose fiber. The cellulose fiber dispersion may be used in the state of dispersion without drying.

  Dispersers used in the above dispersion process include high-powered homomixers, high-pressure homogenizers, ultra-high-pressure homogenizers, ultrasonic dispersion processing, beaters, disk type refiners, conical type refiners, double disk type refiners, grinders, etc. By using a device having a beating ability, it is preferable in that a more efficient and advanced downsizing is possible, and a dispersion can be obtained economically advantageously. As the disperser, for example, a screw mixer, a paddle mixer, a disper mixer, a turbine mixer, or the like may be used.

As a drying method of the cellulose fiber dispersion, for example, when the dispersion medium is water, spray drying, freeze drying, or the like is used. When the dispersion medium is a mixed solution of water and an organic solvent, a drum is used. A drying method using a dryer, a spray drying method using a spray dryer, or the like is used.
The content of the specific cellulose fiber in the dispersion is preferably in the range of 6.0 × 10 ⁻⁵ wt% to 5.0 wt%. When the content of the specific cellulose fiber is within the above range, a sufficient cell growth promoting effect can be obtained.

(4) Addition of other additives In addition to the specific cellulose fiber and dispersion medium, a functional additive can be used as the other component material in the cell growth promoter of the present invention. Examples of the functional additives include thickening accelerators, inorganic salts, organic salts, surfactants, oils, moisturizers, preservatives, organic fine particles, inorganic fine particles, deodorants, fragrances, organic solvents, and the like. can give. These may be used alone or in combination of two or more.

  As the thickening accelerator, carboxyvinyl polymer and alkyl (meth) acrylate copolymer are used. As the (meth) alkyl acrylate copolymer, an alkyl acrylate copolymer, an alkyl methacrylate copolymer, or the like can be used. And in this invention, these thickening promoters are used individually or in combination of 2 or more types.

The inorganic salts are preferably those that can be dissolved / dispersed in water, for example, salts made of alkali metal, alkaline earth metal, transition metal, hydrogen halide, sulfuric acid, carbonic acid, and the like. , KCl, CaCl ₂ , MgCl ₂ , (NH ₄ ) ₂ SO ₄ , Na ₂ CO _{3 and the} like. These may be used alone or in combination of two or more.

  Organic salts are substantially water-soluble by neutralizing hydroxides such as alkali metals and alkaline earth metals, organic amines and carboxyl groups, phosphoric acid groups, sulfonic acid groups, etc. present in the molecule. What is the substance which shows water dispersibility is preferable.

  As the surfactant, those that can be dissolved / dispersed in water are preferable, and examples thereof include sulfonic acid surfactants such as sodium alkylsulfosuccinate, sodium alkylsulfonate, and alkyl sulfate ester salts, and polyoxyethylene alkyl phosphate esters. Nonionic surfactants such as phosphate ester surfactants, alkylene oxide adducts of higher alcohols, and alkylene oxide adducts of alkylarylphenols. These may be used alone or in combination of two or more.

  Examples of oils include silicone oils such as methylpolysiloxane and silicone polyether copolymer, vegetable oils such as olive oil and castor oil, animal oils, lanolin, liquid paraffin, squalane and the like. These may be used alone or in combination of two or more.

  Examples of the humectant include hyaluronic acid, glycerin, 1,3-butylene glycol, sorbitol, dipropylene glycol and the like. These may be used alone or in combination of two or more.

  Examples of the organic fine particles include styrene-butadiene latex, acrylic emulsion, urethane emulsion and the like. These may be used alone or in combination of two or more.

  Examples of the inorganic fine particles include titanium oxide, silica compound, and carbon black. These may be used alone or in combination of two or more.

  Examples of preservatives include methyl paraben and ethyl paraben, and these may be used alone or in combination of two or more.

  Deodorants and fragrances include, for example, D limonene, decyl aldehyde, menthone, pulegone, eugenol, cinnamaldehyde, benzaldehyde, menthol, peppermint oil, lemon oil, orange oil, plants (e.g. Deodorant active ingredients extracted from water, hydrophilic organic solvents, etc. from each organ of black pine, larch, red pine, giraffe, holly mushroom, lilac, guinea pig, fuchsia, Japanese algae, forsythia and the like. These may be used alone or in combination of two or more.

  Examples of the organic solvent include water-soluble alcohols (methanol, ethanol, isopropanol, isobutanol, sec-butanol, tert-butanol, methyl cellosolve, ethyl cellosolve, ethylene glycol, glycerin, etc.), ethers (ethylene glycol) Dimethyl ether, 1,4-dioxane, tetrahydrofuran and the like), ketones (acetone, methyl ethyl ketone), N, N-dimethylformamide, N, N-dimethylacetamide, dimethyl sulfoxide and the like. These may be used alone or in combination of two or more.

  Moreover, the compounding quantity of a functional additive is used by the compounding quantity required in order for the functional additive to express the target effect.

  As described above, the cell growth promoter of the present invention can be obtained by blending a specific cellulose fiber and water, and further, if necessary, a functional additive, followed by a mixing treatment.

  More specifically, examples of the mixing treatment include various homogenizers such as vacuum homomixers, dispersers, propeller mixers, kneaders, various pulverizers, blenders, homogenizers, ultrasonic homogenizers, colloid mills, pebble mills, bead mill pulverizers, high pressure Examples thereof include a mixing process using a homogenizer, an ultrahigh pressure homogenizer, or the like. In addition, although the said mixing process can be performed at normal temperature as stated above, it is also possible to heat as needed, The temperature range is preferably in the range of 5-95 degreeC. More preferably, it is in the range of 10 to 30 ° C.

  The fibroblast proliferation promoter of this embodiment has the effect of promoting the proliferation of the fibroblasts of the skin and prolonging the life. By promoting the growth of fibroblasts, it supports the extracellular matrix produced by fibroblasts, such as collagen that contributes to skin elasticity, hyaluronic acid, a moisturizing component, and collagen fibers, and contributes to skin elasticity. An increase in elastin is expected. It is known that fibroblasts decrease with aging or when the skin is exposed to ultraviolet rays and active oxygen. This also reduces the extracellular matrix such as collagen brought about by fibroblasts, resulting in aging phenomena such as a reduction in the thickness of the dermis, the occurrence of wrinkles and sagging of the skin, the disappearance of the beam, and the loss of moisture retention. When the growth of skin fibroblasts is promoted by the fibroblast proliferation promoter of this embodiment, it is expected to prevent wrinkles and sagging, to prevent the disappearance of beams, and to improve the moisture retention of the skin. It is effective in preventing and improving skin aging. Therefore, the fibroblast proliferation promoter of this embodiment can also be used as an anti-aging or improving agent for skin.

  It is also known that when skin tissue is damaged, fibroblasts migrate and proliferate from the damaged site, and the damaged site is repaired by synthesis of the extracellular matrix. When the proliferation of fibroblasts in the skin is promoted by the fibroblast proliferation promoter of this embodiment, it is expected that damage repair of the skin tissue is promoted and healing is accelerated. Therefore, the fibroblast proliferation promoter of this embodiment can also be used as a skin wound healing promoter.

  As a specific blending form of the fibroblast proliferation promoting agent of the present embodiment, it is applied as a skin external preparation, cosmetics, pharmaceuticals, research reagents, foods and drinks, etc. for the purpose of obtaining the above-mentioned effects. Can do. Among these, the fibroblast proliferation promoter of this embodiment is preferably applied to the skin surface (epidermis) as a skin external preparation or cosmetic. When the fibroblast proliferation promoter of this embodiment is applied as an external preparation for skin, cosmetics, and foods and drinks, in order to distinguish it from conventional products, the above actions / effects such as the promotion of proliferation of skin fibroblasts, wrinkles, etc. It is preferable to indicate that the purpose is to obtain effects such as prevention of sagging, prevention of beam loss, improvement of skin moisture retention, prevention / improvement of aging, and promotion of skin wound healing.

  When applying the fibroblast proliferation promoter of this embodiment to cosmetics, it can manufacture by mix | blending with a cosmetic base material. The form of the cosmetic may be any of emulsion, cream, powder and the like. By applying such cosmetics to the skin, it is possible to obtain a fibroblast proliferation promoting action. Cosmetic bases are generally blended in common with cosmetics. For example, oil, purified water, and alcohol as main components, surfactants, moisturizers, antioxidants, thickeners, anti-seborrheic agents. At least one selected from an agent, a blood circulation promoter, a whitening agent, a pH adjuster, a pigment, a preservative, and a fragrance is appropriately added.

  When the fibroblast growth promoter of this embodiment is applied to a food or drink, the fibroblast growth promoter can be used as a food or drink itself or by blending it with various food materials or beverage materials. The form of the food or drink is not particularly limited, and may be any of liquid, powder, gel, and solid, and the dosage form is any of tablets, capsules, granules, and drinks. May be. Among these, a capsule is preferable because hygroscopicity is suppressed. As said food-drinks, you may mix | blend gelatinizer containing foodstuffs, saccharides, a fragrance | flavor, a sweetener, fats and oils, a base material, an excipient | filler, a food additive, a subsidiary material, a bulking agent etc. suitably as another component.

  When the fibroblast growth promoter of this embodiment is used as a pharmaceutical material or medicine, it can be administered by application to the skin, taken (orally ingested), subcutaneous injection, intravascular administration, transdermal administration, etc. Any administration method can be employed. Although it does not specifically limit as a dosage form, For example, a powder, a powder agent, a granule, a tablet, a capsule, a pill, a suppository, a liquid agent, an injection, etc. are mentioned. Moreover, you may mix | blend an excipient | filler, a base, an emulsifier, a solvent, a stabilizer etc. as an additive. Further, the fibroblast proliferation promoting agent of the present embodiment may be applied as an experimental / research reagent in the form of a fibroblast proliferation promoting reagent. It is preferably used in fields such as elucidation of the mechanism of physiological action related to fibroblasts or research and development such as treatment methods for various symptoms.

Next, examples of the present invention will be described. However, the present invention is not limited to these examples.
<Test Example 1: Cell proliferation promoting effect confirmation test of cellulose fiber>
In the test for confirming the fibroblast proliferation promoting effect of the cellulose fiber of this test example, pure water was added to an aqueous dispersion of cellulose fiber synthesized based on the following synthesis example. K. A 10 wt% diluted solution (solid content concentration: 0.2 wt%) obtained by stirring at 8000 rpm for 10 minutes with a homomixer (manufactured by PRIMIX) was used.
Synthesis example:
To 2 g of softwood pulp, 150 ml of water, 0.25 g of sodium bromide and 0.025 g of TEMPO were added and dispersed with sufficient stirring, and then a 13 wt% sodium hypochlorite aqueous solution (co-oxidant) was added. The reaction was started by adding sodium hypochlorite to 6.5 mmol / g with respect to 1.0 g of the pulp. Since the pH decreased with the progress of the reaction, the reaction was continued until no change in pH was observed while dropping a 0.5N aqueous sodium hydroxide solution so that the pH was maintained at 10-11 (reaction time: 120 minutes). ). After completion of the reaction, 0.1N hydrochloric acid was added to adjust the pH to 7.0, and purification was repeated by filtration and washing to obtain cellulose fibers having oxidized fiber surfaces. The cellulose group had a carboxyl group amount and a number average fiber diameter of 1.83 mmol / g carboxyl group and a number average fiber diameter of 6 nm. Further, it was confirmed by wide-angle X-ray diffraction image measurement that the obtained cellulose fiber had an I-type crystal structure.
Modified human normal skin fibroblasts (manufactured by DS Pharma, Cell System-Fb Cells) in the logarithmic growth phase, 0.5% fetal bovine serum (FBS), Dulbecco's modification to 96 cells in a 96-well plate The seeds were suspended in Eagle's medium (DMEM) and cultivated for 1 day.

As an example of the cellulose fiber of the present invention, 0.03125% by weight of each of the above cellulose fiber aqueous dispersions having a solid content concentration of 0.2% by weight (cellulose fiber concentration: 6.25 × 10 ⁻⁵ wt. %), 0.0625 wt% (cellulose fiber concentration 1.25 × 10 ⁻⁴ wt%), 0.125 wt% (cellulose fiber concentration 2.5 × 10 ⁻⁴ wt%), 0.25 wt% (cellulose) Fiber concentration 5.0 × 10 ⁻⁴ wt%), 0.5 wt% (cellulose fiber concentration 1.0 × 10 ⁻³ wt%), 1 wt% (cellulose fiber concentration 2.0 × 10 ⁻³ wt%) , 2% by weight (cellulose fiber concentration 4.0 × 10 ⁻³ wt%) was diluted with 0.5% FBS and DMEM and added to each well. Moreover, FBS and DMEM were added to positive control (PC) so that it might become 10% FBS and DMEM, without adding the said aqueous dispersion of a cellulose fiber. The negative control (NC) was adjusted to 0.5% FBS, DMEM without adding the above aqueous dispersion of cellulose fibers. These samples were further cultured.

  Coloring reagent CCK-8 for cell proliferation measurement using water-soluble tetrazolium salt WST-8 as a coloring reagent before adding the aqueous dispersion of cellulose fiber, after culturing for 1 day, after culturing for 2 days, and after culturing for 3 days. (Dojindo Laboratories Co., Ltd.) was diluted 20-fold with DMEM, added at 100 μL / well, and subjected to a color reaction for 2 hours, and the absorbance at 450 nm was measured with a microplate reader. For the positive control (PC) and the negative control (NC), the absorbance at 450 nm was measured in the same manner.

The results are shown in FIG. 1 and FIG.
FIG. 1 is a graph showing the rate of increase in the number of cells in each sample after 1, 2, and 3 days of culture, where the number of cells on day 0 of culture (before addition of the aqueous dispersion of cellulose fibers) is defined as 100. FIG. 2 is a graph showing the number of cells in each sample after 3 days of culture, and the number of cells on the third day of the negative control (NC) is 100%.
From the results of FIGS. 1 and 2, when the concentration of the aqueous dispersion of the cellulose fiber is 0.03125% by weight or more with a solid content concentration of 0.2% by weight, that is, the cellulose fiber concentration is 6.25 × 10 ⁻⁵ % by weight. At the above time, cell proliferation was promoted.

<Test Example 2: Cell migration promoting effect confirmation test of cellulose fiber>
As an example of the cellulose fiber of the present invention, the above-described aqueous dispersion of cellulose fiber having a solid content concentration of 0.2% by weight prepared in the synthesis example of Test Example 1 was used to promote the fibroblast migration promoting effect of the composition. Tested.
When normal human dermal fibroblasts were cultured in a culture dish and became confluent, they were injured with a needle tip. 3 wt% (cellulose fiber concentration 6.0 × 10 ⁻³ wt%), 0.75 wt% (cellulose fiber concentration 1.5 ×) 500 μL each of a solution diluted with DMEM medium so as to have a concentration of 10 ⁻³ wt%, positive control PC (10% FBS, DMEM medium), and negative control NC (DMEM medium) were added and cultured for 7 hours. .
Then, using the area of the wound immediately after the wound and the value of the area of the wound after culturing for 7 hours, the change rate indicating the ratio of the area where the wound was closed was calculated and plotted on a graph.
That is, the rate of change is {(the area of the wound immediately after being scratched (μm ² ) −the area of the wound after being cultured for 7 hours (μm ² )) / the area of the wound immediately after being scratched (μm ² )} X100 (%) was calculated.

The results are shown in FIGS.
FIG. 3 is a photograph showing the appearance of the wound before and after culturing after scratching in this test example, and FIG. 4 is a graph of the calculated change rate.
From the results of FIGS. 3 and 4, it was found that the cellulose nanofiber composition of this example promoted migration of fibroblasts.

Claims (4)

A cell growth promoter,
Cellulose fiber as an active ingredient,
The cellulose fiber has a number average fiber diameter of 2 to 150 nm. The cellulose has a cellulose I-type crystal structure, and a hydroxyl group at the C6 position of each glucose unit in the cellulose molecule is selectively oxidized to form a carboxyl. A cell growth promoter characterized by being modified into a group, whereby the carboxyl group has a ratio of 0.6 to 2.5 mmol / g.   The cell growth promoter according to claim 1, wherein the cellulose fiber is oxidized using a co-oxidant in the presence of an N-oxyl compound. The cell growth promoter is a dispersion in which the cellulose fibers are dispersed in an aqueous solvent,
The cell growth promoter according to claim 1 or 2, wherein a concentration of the cellulose fiber in the cell growth promoter is 6.0 x ^10-5 wt% to 5.0 wt%.   The cell growth promoter according to any one of claims 1 to 3, which is a fibroblast growth promoter.