JP2020094013A - Cell activator - Google Patents

Abstract

To provide a cell activator, and to effectively utilize a culture medium that has been used for culturing Euglena.SOLUTION: A cell activator contains a culture medium after culturing of Euglena and/or a concentrate thereof.SELECTED DRAWING: None

Description

The present invention relates to a cell activating agent and the like.

Due to various factors such as aging, ultraviolet rays, dryness, stress, etc., the flexibility and moisturizing function of the skin decrease, and when this progresses, skin aging symptoms (decrease in tension, luster, roughness, wrinkles, dullness, etc.) may occur. Manifest. Various cell activating agents have been proposed to suppress such a phenomenon at the cellular level. If the cells are activated and the turnover of skin cells is promoted and the extracellular matrix is increased, the above phenomenon can be suppressed. For example, Patent Document 1 reports that glyceric acid is used as a cell activating agent.

Euglena is a microalgae belonging to the Euglena genus (= Euglena genus) and is used as a food material. In addition, the Euglena extract is also applied to the skin (Patent Document 2).

JP 2016-003209 JP Special Table 2008-526954 Publication

An object of the present invention is to provide a cell activating agent.

Further, the use of Euglena is limited to the use of Euglena itself and its extract, and the cultured medium generated when preparing these has been discarded. The present inventor has paid attention to the point of effectively utilizing this waste liquid during the progress of research. Therefore, the present invention also aims to effectively utilize the cultured medium of Euglena.

As a result of earnest studies in view of the above problems, the present inventor has found that the culture solution after Euglena culture and/or its concentrate has a cell activating effect. As a result of further research based on this finding, the present invention has been completed.

That is, the present invention includes the following aspects.

Item 1. A cell activating agent containing a culture solution after Euglena culture and/or a concentrate thereof.

Item 2. Item 2. The cell activating agent according to Item 1, wherein the Euglena is Euglena gracilis.

Item 3. Item 3. The cell activating agent according to Item 1 or 2, wherein the Euglena is Euglena gracilis EOD-1 strain (Accession No. FERM BP-11530).

Item 4. Item 4. The cell activating agent according to any one of Items 1 to 3, wherein the cells are skin cells.

Item 5. Item 5. The cell activating agent according to any one of Items 1 to 4, which is a composition for external use.

Item 6. Item 6. The cell activating agent according to any one of Items 1 to 5, which is a cosmetic, a cosmetic additive, or a medicine.

According to the present invention, a cell activating agent can be provided. This cell activating agent uses a culture solution after Euglena culture and/or a concentrate thereof as an active ingredient, and effectively utilizes a Euglena cultured medium.

The result of a cell activation test (test example 1) is shown. The vertical axis represents the relative number of viable cells when the comparison target is 1. The horizontal axis represents the final concentration in the test well of the culture solution after Euglena culture (Production Example 1) or the medium for Euglena culture (Comparative Production Example 1). Regarding the p-value obtained by comparison with 0% (comparison target) by Dunnett's multiple comparison test, * is shown when p <0.05, and ** is shown when p <0.01.

In this specification, the expressions "containing" and "including" include the concepts of "containing", "including", "consisting essentially of" and "consisting solely of".

In one aspect thereof, the present invention relates to a cell activating agent (also referred to herein as "agent of the present invention") containing a culture solution after Euglena culture and/or a concentrate thereof. This will be described below.

1. Euglena culture medium and/or its concentrate Euglena are microalgae belonging to Euglena (genus Euglena), and are not particularly limited as long as they are. As Euglena, specifically, for example, Euglena gracilis (Euglena gracilis), Euglena longa, Euglena caudata, Euglena oxyuris, Euglena tripteris, Euglena proxima, Euglena viridis, Euglena sociabilis, Euglena ehrenbergii, Euglena deses, Euglena pisciformis, Euglena spirogyra, Examples include Euglena acus , Euglena geniculata , Euglena intermedia , Euglena mutabilis , Euglena sanguinea , Euglena stellata , Euglena terricola , Euglena klebsi , Euglena rubra , Euglena cyclopicola . Among these, Euglena gracilis is preferable, and Euglena gracilis EOD-1 strain is more preferable from the viewpoint that the effect of the present invention can be exhibited more reliably. Under the provisions of the Budapest Treaty, deposit number FERM BP-11530 at the Japan Patent Organism Depositary {NITE-IPOD (Zip Code 292-0818 Kazusa Kamasa 2-5-8 120, Kisarazu City, Chiba Prefecture, Japan)} International Deposited].

The content of paramylon in the dry state of euglena is, for example, 50% or more, preferably 60% or more, more preferably 70% or more.

Euglena may be a single species or a combination of two or more species.

The culture solution after Euglena culture and/or its concentrate is a culture solution obtained by culturing Euglena, and is not particularly limited as long as it is. It is considered that the culture solution after the Euglena culture contains a group of components secreted from living cells of Euglena during the culture process. On the other hand, the Euglena extract utilizes dead cells whose cell membrane is broken, and contains most of its internal components. Therefore, the culture solution after the Euglena culture is different from the Euglena extract in its component composition.

The culture of Euglena can be performed by a method including a step of culturing Euglena contained in a liquid (culture step). The culturing step can be performed, for example, according to a known method (for example, the method described in Japanese Patent No. 5883532). In the culturing step, typically, a euglena genus microalga is cultivated under aerobic conditions while stirring a liquid (medium) containing water, euglena, and nutrients that can be used by euglena.

As the nutrients, sugars (glucose (glucose), fructose (fructose) and other monosaccharides, or sucrose (sucrose), maltose (maltose) and other disaccharides), minerals (for example, sodium, potassium, magnesium, calcium, Iron, zinc, molybdenum, copper, phosphorus, nitrogen, sulfur, or boron), vitamin Bs (for example, vitamin B1 (thiamine), vitamin B2 (riboflavin), niacin, pantothenic acid, vitamin B6 (pyridoxine, pyridoxal, or Pyridoxamine), vitamin B12 (cyanocobalamin), folic acid, biotin, etc.) and the like. The concentration of nutrients in the culture solution is not particularly limited as long as it is a concentration at which Euglena can survive and proliferate.

The light conditions in the culturing step are not particularly limited, and the culturing step may be performed under either bright or dark conditions. When culturing in heterotrophic culture, it is cultivated under dark conditions. As the light condition, normal light intensity for growing algae can be adopted. The dark conditions include, for example, less than 10 μmol/m ² /s, and preferably complete dark conditions where no light is applied.

The culture temperature in the culture step is not particularly limited as long as it is a temperature at which Euglena can grow. As the culture temperature (temperature of the culture solution), for example, 20°C to 35°C is adopted.

The pH of the liquid in the culturing step is not particularly limited as long as it is a pH at which Euglena can grow. The pH at which Euglena can grow is, for example, 3.0 to 5.5.

As described above, the culture medium after euglena culture contains a group of components secreted from euglena during the culture step, and these are considered to have a cell activating effect. The culturing period in the culturing step is not particularly limited as long as these components are secreted, for example, 12 to 240 hours, preferably 24 to 144 hours, more preferably 48 to 96 hours, further preferably 60. ~ 84 hours.

The culture solution after the culturing step can be used as it is as a culturing solution after the Euglena culture, or insoluble components (for example, euglena cell bodies, fragments of euglena cell bodies, and mucus secreted from euglena) from the culture solution after the culturing step. The Euglena culture supernatant obtained by reducing or removing qualitative polysaccharides) can also be used as a culture solution after Euglena culture. As the culture solution after Euglena culture, Euglena culture supernatant is preferable.

The method for reducing or removing the insoluble matter is not particularly limited, and a known method can be adopted. Examples of the method include centrifugation, gravity separation, filtration and the like. These methods may be used alone or in combination of two or more.

The condition of centrifugation is not particularly limited as long as it is a condition that an insoluble component in the culture solution after the culture step can be precipitated. For example, conditions of 1000 to 5000 g (preferably 2000 to 4000 g) for 1 to 30 minutes (preferably 2 to 10 minutes) can be mentioned.

The filtration conditions are not particularly limited as long as the conditions are such that the insoluble components in the culture solution after the culture step can be reduced or removed. For example, a filter having a pore size of about 0.1 to 1 μm (preferably 0.1 to 0.5 μm) can be used.

The concentrate of the culture solution after euglena culture can be obtained by concentrating (reducing or removing water) the culture solution after euglena culture by an appropriate method (for example, drying).

2. Use Since the culture solution after Euglena culture and/or its concentrate has a cell activating effect, it can be used as an active ingredient of a cell activating agent. It can also be used as an active ingredient for various uses based on the cell activation action, for example, a cell growth promoter, a cell senescence suppressor, an extracellular matrix secretion promoter, a cell turnover promoter and the like.

The target cells of the agent of the present invention are not particularly limited. A typical example of the target cells is skin cells. Examples of skin cells include dermal cells such as fibroblasts; epidermal cells such as granule cells, spinous cells, basal cells and the like.

The agent of the present invention is used in various fields as, for example, cosmetics, cosmetic additives, pharmaceuticals, food compositions (including health foods, health promoters, dietary supplements (supplements etc.)), food additives, feeds, etc. be able to. The agent of the present invention is preferably an external composition.

The form of the agent of the present invention is not particularly limited and may be a form usually used in each application depending on the application.

As the form of the agent of the present invention, when the application is cosmetics, for example, emulsion, cosmetic liquid, face cream, hand cream, lotion, body soap, shampoo, rinse, cosmetic gel, pack, foundation, lip balm, face wash Agents and the like.

As the form of the agent of the present invention, when the use is a medicine, for example, an ointment, an external liquid agent (a liniment agent, a lotion agent, etc.), a spray agent (an external aerosol agent, a pump spray agent, etc.), a cream agent, a gel agent , Patches (plasters, tapes such as plasters (reservoir type, matrix type, etc.), poultices, patches, microneedles, etc.), eye drops, eye ointments, nasal drops, suppositories, semi-rectal Formulations suitable for parenteral ingestion such as solid dosage forms and enema preparations (especially external preparation forms); tablets (including disintegrating buccal tablets, chewable tablets, effervescent tablets, troches, jelly-like drops), Formulations suitable for oral ingestion such as pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups) and jellies ( Oral formulation form) is preferred, and external formulation form is preferred.

As the form of the agent of the present invention, when the use is an additive, a health promoting agent, a dietary supplement (supplement, etc.), for example, tablets (intraorally disintegrating tablet, chewable tablet, effervescent tablet, lozenge, jelly) Pills, pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including suspensions and syrups), and jellies.

As the form of the agent of the present invention, when the application is a food composition, liquid, gel-like or solid food, for example, juice, soft drink, tea, soup, soy milk beverage, salad oil, dressing, yogurt, jelly , Pudding, sprinkles, baby milk powder, cake mix, dairy products (eg, powder, liquid, gel, solid, etc.), bread, confectionery (eg, cookies, etc.) and the like.

The agent of the present invention may further contain other components, if necessary. Other components are components that can be incorporated into cosmetics, cosmetic additives, medicines, food compositions (including health foods, health promoters, dietary supplements (supplements, etc.)), food additives, feeds, etc. As long as it is not particularly limited, for example, bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, colorants, Examples include fragrances and chelating agents.

The content ratio of the culture solution after Euglena culture and/or the concentrate thereof in the agent of the present invention depends on the application, the mode of use, the state of the application target, and is not limited. For example, it can be 0.0001 to 100% by mass in terms of culture medium after Euglena culture. The lower limit value is, for example, 0.001, 0.01, 0.1, 1, 2, 5, 7, 10% by mass. The upper limit is, for example, 70, 50, 40, 30, 25, 15% by mass.

The application amount (eg, administration, ingestion, inoculation, etc.) of the agent of the present invention is not particularly limited as long as it is an effective amount that exerts a cell activating effect on target cells, and is usually a culture solution component after Euglena culture. The dry weight of is generally 0.1-10000 mg/kg body weight per day. The above-mentioned applied amount is preferably applied once or more times a day (for example, 1 to 3 times), and may be appropriately increased or decreased depending on age, disease state and symptoms.

Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.

Production example 1
Euglena gracilis EOD-1 strain (NITE-IPOD, National Institute for Product Evaluation Technology, National Institute for Product Evaluation Technology) was cultured in a medium based on Hutner medium to obtain a culture solution. This culture solution was centrifuged. (3000 g, 5 minutes), and the supernatant was recovered, and the obtained supernatant was filtered (Nalgene Rapid-Flow PES filter unit 0.2 μm, 75 mm, 500 mL (product number: 566-0020)) and the filtrate was collected. Insoluble components (for example, Euglena cell bodies, fragments of Euglena cell bodies, and viscous polysaccharides secreted from Euglena) were removed by centrifugation and filtration. It was used as a post-culture liquid in the following test examples.The post-Euglena culture liquid culture medium was stored refrigerated under light shielding until use.

Comparative Production Example 1
A medium based on the Hutner medium described in Production Example 1 (one that has not been subjected to culture (not mixed with Euglena)) was prepared. This was centrifuged (3000 g, 5 minutes) and the supernatant was collected. The resulting supernatant was filtered (Nalgene Rapid-Flow PES filter unit 0.2 μm, 75 mm, 500 mL (product number: 566-0020)) to collect the filtrate. It was kept refrigerated under light shielding until use.

Test example 1. Cell activation test After Euglena culture, the cell activation effect of the culture solution was examined. Euglena culture medium was used as a control. Specifically, it was performed as follows.

<Materials, equipment, etc.>
Human normal skin fibroblasts RIKEN Cell Bank RCB0589
MEMα: GIBCO 12571063
FBS Gibco Performance Grade 26140079
Cell Counting Kit-8 Dojindo Laboratories Product Code: CK04
Cell culture multi-dish 96-well Thermo Fisher Part number 167008
0.25w/v% trypsin solution 100mL Wako 201-18841
PBS (-) 500ml Wako 166-23555

<Method>
Human normal skin fibroblasts were pre-cultured in about 17 ml of medium 1 (MEMα+15% FBS) at 37° C. and 5% CO ₂ using a T75 flask as a culture container. After confirming that the cells were 80 to 90% confluent, the medium was removed. After adding 17 ml of PBS to make the cells familiar with the cells, PBS was removed. The cells were detached by adding 6 ml of 0.25% trypsin/PBS and incubating at 37° C. under 5% CO ₂ for 5 minutes. Trypsin was inactivated by adding 9 ml of medium 1. The supernatant was removed by centrifugation (300 g, 5 minutes). The cells were suspended in 17 ml of Medium 2 (MEMα+0.5% FBS). The supernatant was removed by centrifugation (300 g, 5 minutes). The cells were suspended in 3 ml of medium 2. The number of cells was counted using a hemocytometer and diluted with Medium 2 to 3.3×10 ⁴ cells/ml. 75 μl of the obtained cell suspension was dispensed into each well of a 96-well plate. A culture solution after Euglena culture (Production Example 1) or a medium for Euglena culture (Comparative Production Example 1) was diluted with Medium 2 to prepare 40, 20, and 8%, and 25 μl was added to each well of a 96-well plate. (The final concentration of the culture solution after Euglena culture (Production Example 1) or the medium for Euglena culture (Comparative Production Example 1) in each well is 10, 5, or 2%). As a comparison target, a well to which only 25 μl of medium 2 was added was prepared (post-culture medium and culture medium were not included). It was cultured for 48 hours under the conditions of 37° C. and 5% CO ₂ .

After the culture was completed, live cells were measured using Cell Counting Kt-8. The measurement followed the attached instruction. The specific method is as follows. 10 μl of Cell Counting Kit-8 solution was added to each well of the 96-well plate, and incubated at 37° C. under 5% CO ₂ for 4 hours. The absorbance at 450 nm/600 nm was measured with a microplate reader. Three wells were tested under the same conditions, the average value and the standard deviation were calculated, and the absorbance of the comparison target was set to 1, and the numbers of viable cells were relatively compared. The p-value was calculated by comparison with 0% (comparison target) by Dunnett's multiple comparison test.

<Results>
The results are shown in Figure 1. It was shown that the culture medium after Euglena culture increased the number of viable cells. From this, it was found that the culture solution after the Euglena culture has a cell activation effect.

Claims (6)

A cell activating agent containing a culture solution after Euglena culture and/or a concentrate thereof. The cell activating agent according to claim 1, wherein the Euglena is Euglena gracilis. The cell activating agent according to claim 1 or 2, wherein the Euglena is Euglena gracilis EOD-1 strain (accession number FERM BP-11530). The cell activating agent according to claim 1, wherein the cells are skin cells. The cell activating agent according to any one of claims 1 to 4, which is a composition for external use. The cell activating agent according to any one of claims 1 to 5, which is a cosmetic, a cosmetic additive, or a medicine.