WO2005059114A1 - Fat composition for promoting animal cell growth, medium for culturing animal cells containing the composition and external skin preparation - Google Patents

Abstract

It is intended to provide a fat composition which is less expensive and highly stable in qualities and has a function of promoting animal cell growth, a medium for culturing animal cells containing this fat composition, and an external skin preparation having an effect of ameliorating or healing skin symptoms. Namely, a fat composition for promoting animal cell growth which is a fat composition comprising a triglyceride, a diglyceride, a monoglyceride and a free fatty acid and containing at least one member selected from Ϝ-linolenic acid and Ϝ-linolenic acid glyceride or an external skin preparation, and a medium for culturing animal cells containing the fat composition.

Description

 Specification

 Animal cell growth promoter oil composition, animal cell culture medium containing the composition, and skin external preparation composition

 Technical field

[0001] The present invention relates to an animal cell growth promoting oil / fat composition comprising _{{monolinolenic} acid (hereinafter, abbreviated as GLA)} 7-linolenic acid glyceride having animal cell growth promoting ability, and The present invention relates to a medium for culturing animal cells comprising the oil / fat composition.

 In addition, the present invention relates to a skin external preparation composition comprising, as an active ingredient, a fat or oil composition containing γ-linolenic acid or γ-linolenic acid dalyseride having an ability to promote animal cell growth.

 Background art

 [0002] In recent years, along with the development of molecular biology, the necessity of animal cell culture has been markedly increased. Animal cell culture technology is widely used, for example, for cell fusion, production of monoclonal antibodies, production of vaccines, and the like. Is a biological technology.

 Animal cells do not grow in a medium containing only nutrients such as amino acids, sugars, salts, etc. In order to grow animal cells, a cell growth agent other than mere nutrients is required.

 Conventionally, various sera such as fetal calf serum have been used as such a cell growth agent. However, since there are multiple cell growth factors in serum, and at present most of the factors are unknown components, the content of those factors cannot be measured. For this reason, the animal cell culture medium using the above-mentioned serum has a problem that cell growth activity varies from medium to medium. In addition, since the activity of cell growth factor in serum varies depending on the individual animal to be collected, there is a problem that the quality of serum is not constant.

On the other hand, in addition to the serum, various animal cell promoting agents have been developed. For example, 5-aminolevulinic acid (for example, see Patent Document 1), a substance produced by the genus Rhizopus (RU substance) (for example, see Patent Document 2), 5-hydroxy-2,7-dimethoxy 6-methyl-1,4-naphthoquinone (See, for example, Patent Document 3), Panax ginseng extract (see, for example, Patent Document 4), Animal cell promoters such as toglycogen (for fibroblasts) (for example, see Patent Document 5) and sugar esters of sugars (monosaccharides and disaccharides) and fatty acids (for example, see Patent Document 6) have been developed. However, these animal cell promoters have problems such as difficulty in production, high production cost, and unstable quality, and the development of an inexpensive animal cell promoter with excellent quality stability is expected. It is rare.

 In the field of external preparations for skin, many studies have been conducted to activate skin cells and activate skin functions themselves to improve skin symptoms and to exhibit an anti-inflammatory effect or a wound healing effect. Conventionally, various substances such as hormone preparations, vitamin preparations, crude drug extracts such as γ-oryzanol and saponin, plant lectins, mushroom extracts, and protein derived from animals have been used as powerful external preparations for the skin.

 Among these substances, retinol polyhydroxyacid is effective in repairing wrinkles as a substance that has the effect of improving skin aging symptoms such as wrinkles and a substance that prevents skin aging and inflammatory effects due to sunlight and ultraviolet rays. It is known that However, among the α-hydroxy acids, dalicholic acid and lactic acid, which have particularly effective effects, are lipophilic, and if the compounding amount is poor in percutaneous absorption, undesired side effects such as skin irritation may occur. There were also things. In addition, retinol is easily oxidized when exposed to air, and is usually used as an ester with fatty acids S. Since this fatty acid ester is fat-soluble, it is difficult to mix it with a water-soluble base material. there were.

The healing of wounds (surgical incisions, wounds or ulcers in the gastrointestinal tract, abrasions, lacerations, amputations, so-called bed sores, sores, infections and other damage to superficial tissues) involves cell proliferation and epithelial tissue. It is thought that an agent that stimulates or promotes the process of differentiation and proliferation of cells involved in the healing process of a wound, which depends on the formation of cells, is effective. Extracts such as aloe, antibiotics, anti-inflammatory drugs, kallikrein, adenine, nicotinic acid, allantoin, vitamin A, zinc, and cAMP derivatives as active ingredients that exhibit wound healing effects (see, for example, Patent Document 7) In addition to the above, attempts have been made to use the above-mentioned cell growth factor (b-EGF) (for example, see Patent Document 8). However, the above-mentioned active ingredient may not be able to obtain a sufficient effect, and there are problems that production is difficult, the production cost is high, and the active ingredient is chemically unstable. Excellent, has the effect of improving or healing skin symptoms There is a need for a skin external preparation that does.

 [0003] Patent Document 1: JP-A-5-49472

 Patent Document 2: JP-A-5-97685

 Patent Document 3: JP-A-5-137569

 Patent Document 4: JP-A-6-239759

 Patent Document 5: JP-A-11-255657

 Patent Document 6: JP-A-2003-52366

 Patent Document 7: JP-A-63-107935

 Patent Document 8: JP-A-3-106823

 Disclosure of the invention

 Problems to be solved by the invention

[0004] The present invention has been made in view of the above circumstances, and provides an oil / fat composition which is inexpensive, has excellent quality stability, and has an ability to promote the growth of animal cells, and a medium for culturing animal cells containing the oil / fat composition. It is intended for that purpose.

 Another object of the present invention is to provide a skin external preparation composition which is inexpensive, has excellent quality stability, and has an effect of improving or healing skin symptoms.

 Means for solving the problem

[0005] The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have found that the above object can be achieved by a fat or oil composition containing a specific substance having an animal cell growth promoting ability. I found it. The present invention has been completed based on powerful knowledge.

That is, the present invention relates to an oil / fat composition containing triglyceride, diglyceride, monoglyceride and free fatty acid, wherein the oil / fat composition contains one or more selected from γ-linolenic acid and _{レ ン -linolenic} acid glyceride. Is provided.

 The present invention also provides an animal cell culture medium containing the animal cell growth promoter oil / fat composition.

Furthermore, the present invention is a triglyceride, diglyceride, a fat composition comprising a monoglyceride and free fatty acids, _I - linolenic acid and _I - to provide a skin external composition containing one or more upper selected from linolenic acid glyceride Is the thing The invention's effect

 [0006] According to the present invention, an animal cell growth promoter oil / fat composition which is inexpensive and has excellent quality stability, and an animal cell culture medium containing the animal cell growth promoter oil / fat composition can be obtained. . Further, a skin external preparation composition which is inexpensive, has excellent quality stability, and has an effect of improving or healing skin symptoms can be obtained.

 BEST MODE FOR CARRYING OUT THE INVENTION

 [0007] The fatty acids constituting triglycerides, diglycerides, and monoglycerides which are components of the animal cell growth promoter oil / fat composition or skin external preparation composition of the present invention are not limited, for example, saturated fatty acids having 2 to 24 carbon atoms and unsaturated fatty acids. Fatty acids. Preferred fatty acids include caprylic acid, capric acid, myristic acid, myristoleic acid, pentadecenoic acid, palmitic acid, panolemitrenic acid, stearic acid, arachidic acid, behenic acid, ligrenolic acid, serotinic acid, oleic acid, and linoleic acid. Monooleic acid, monolinolenic acid, γ-linolenic acid, octadecatetraenoic acid, eicosenoic acid, eicosadic acid, eicosatrienoic acid, eicosatetraenoic acid, arachidonic acid, eicosapentaenoic acid, docosenoic acid, Docosagenic acid, docosapentaenoic acid, docosahexaenoic acid and the like.

 Glyceride is not particularly limited, and is preferably a plant or microbial-derived glyceride, because it can be used in a chemically synthesized product, a plant or animal-derived, or a microbial-derived.

 The content ratio of triglyceride, diglyceride, and monoglyceride contained in the animal cell growth promoter oil composition or the skin external preparation composition of the present invention is based on the total amount of glyceride from the viewpoint of preventing variation in the animal cell growth effect. , Respectively, preferably 10 to 79% by mass, 20 to 70% by mass, and 1 to 70% by mass. The content of triglyceride, diglyceride and monoglyceride is more preferably 10 to 70% by mass, 25 to 60% by mass and 3 to 65% by mass, respectively, based on the total amount of glyceride. Most preferably, they are 10-65% by mass, 30-60% by mass and 5-60% by mass, respectively, based on the total amount of glyceride.

Glycerides having such a composition can be produced by a known method. For example, it is produced by a method of inducing triglyceride type fats and oils into diglycerides and monoglycerides using a chemical catalyst or an enzyme catalyst (for example, see JP-A-2000-236888 and JP-A-2000-333688). The ability to do S.

 [0008] GLA, which is an active ingredient of the oil / fat composition for animal cell growth promoter or the external preparation for skin of the present invention, may exist not only as a free fatty acid but also as an ester. "GLA content" refers to a free fatty acid obtained by hydrolyzing glyceride from all GLA derivatives in the oil / fat composition of the present invention, a fat / oil composition for animal cell growth promoter, or an external preparation for skin. It is calculated from the total free fatty acids present in the composition. For example, when GLA is present as a ester, the amount of free GLA obtained by hydrolyzing this ester with kafunka may be measured.

 The content of GLA in the animal cell growth promoter oil / fat composition may be appropriately adjusted according to the type of animal cell to be subjected to growth promotion. Generally, from the viewpoint of preventing variation in the effect of growing animal cells due to differences in animal cell types, the amount is preferably 5% by mass or more based on the total amount of fatty acids and free fatty acids constituting glyceride. It is more preferably at least 10% by mass, and even more preferably at least 20% by mass. Although there is no particular upper limit to the content of GLA, it is preferably 98% by mass or less from the economical point of view, even if the content exceeds 98% by mass, even if the effect of increasing animal cell proliferation is not further improved.

 Further, the content of GLA in the skin external preparation composition may be appropriately adjusted according to the dosage form and type of the skin external preparation composition. In general, the content is preferably 5% by mass or more based on the total amount of fatty acids and free fatty acids constituting glyceride, from the viewpoint of preventing the healing effect from being varied depending on the dosage form and the intended use. It is more preferably at least 10% by mass, and even more preferably at least 20% by mass. Although there is no particular upper limit to the content of GLA, it is preferably 98% by mass or less from the economical viewpoint, even if the content exceeds 98% by mass, the healing effect is not further improved.

 GLA can also be obtained by chemically synthesizing, plant seeds such as borage, evening primrose, and saxifraga, algae such as Spirulina, Cunninghamella, Mortierella, and Mucor. Microbial power of a genus or the like can also be obtained. When the obtained GLA is a derivative, it may be subjected to hydrolysis treatment or transesterification treatment which may be used as it is.

[0009] The animal cell growth promoting oil / fat composition or skin external preparation composition of the present invention is produced by mixing glyceride and GLA oil / fat at the above-mentioned predetermined ratio by a known mixing method. You can. Further, the animal cell growth promoting oil or fat composition or the skin external preparation composition of the present invention extracts a GLA-containing microbial fat inside a microorganism or a microbial diluting fat from a GLA-containing microorganism, and converts the GLA-containing fat or oil into It can be obtained by hydrolysis or transesterification.

 The GLA-containing fats and oils can be obtained by culturing a microorganism capable of producing GLA-containing fats and oils by a conventional method. As described above, examples of the microorganism having the ability to produce GLA-containing fats and oils include various microorganisms such as filamentous fungi, yeast, and algae. For example, microorganisms having the ability to produce gamma-linolenic acid-containing fats and oils include microorganisms belonging to the genus Mortierella described in JP-A-60-168391 and JP-A-63-283589. And the like, and microorganisms belonging to the genus Rhizopus described in JP-A-63-133994 and the like. More specifically, as a microorganism belonging to the genus Mortierella, for example,

'Ancrispora (Mortierella ramaniana var.angrispora) IF08187 and the like. Examples of the microorganisms belonging to the genus Mucor include, for example, Mucor circinelloides HUT1121 (FERM P-9359) @ Mucor * Javanitas (Mucor javanicus) HUTl 162 (FERM P-9360).

 The medium for culturing the microorganism may be a carbon source, a nitrogen source, inorganic salts, and, if necessary, an amino acid suitable for the growth of the microorganism, as long as the microorganism can grow well and produce a desired lipid. What contains the component of is used. As the carbon source, saccharides such as darcos, starch, molasses, and organic acids and sodium acetate can be used, and saccharides such as gnoleose are particularly preferable. Examples of the nitrogen source include ammonium salt, yeast extract, corn steep liquor, peptone, and the like. Examples of the inorganic salts include magnesium salts, calcium salts, phosphates, iron salts, and copper salts.

In culturing, if the total amount of carbon source, nitrogen source or phosphate is added to the medium from the beginning, it may adversely affect the growth of microorganisms. , Which can improve the productivity of γ-linolenic acid, Other culturing conditions, such as temperature and culturing time, are preferably set in consideration of the medium composition, the target content of γ-linolenic acid, and the productivity of lipids. The temperature is usually about 20 to 35 ° C, preferably 25 to 30 ° C, and ρΗ is usually about 3 to 7, preferably 3.5 to 6.5, usually about 72 to 240 hours, preferably about 96 to 168. It's time to go.

[0011] Since GLA-containing lipids are usually accumulated in microbial cells, the culture solution is subjected to solid-liquid separation by a conventional method to obtain cells containing GLA-containing lipids. Depending on the use of GLA, these cells can be used as they are, but in order to further extract and purify fats and oils, the bacteria are obtained by the methods described in the Bligh & Dyer method, the Folich method, and JP-A-57-144986. By crushing the body and extracting the solvent, the desired GLA-containing fats and oils can be obtained.

 The GLA-containing fat or oil obtained as described above is subjected to hydrolysis treatment or transesterification treatment by a known method to obtain the animal cell growth promoter fat or oil composition or the external skin composition of the present invention. it can. When the animal cell growth promoter oil / fat composition or skin external preparation composition of the present invention is obtained by such a method, the content ratio of GLA in the animal cell growth promoter oil / fat composition or skin external preparation composition is determined as follows. A part of the accelerator or fat composition or the external composition for skin is determined, and after methylesterification, determined by gas chromatography (see JP-A-57-144986).

[0012] The animal cell growth promoter oil / fat composition of the present invention may contain other components as long as they do not inhibit the effects of the present invention. Other components include, for example, the aforementioned fatty acids other than GLA. When a plant-derived or microorganism-derived glyceride GLA is used, other components derived from a plant or a microorganism may be contained. The animal cell growth promoter oil composition of the present invention comprises an adhesion-dependent cell such as epidermal keratinocytes, skin fibroblasts, melanocytes, small intestinal epithelial cells, human cervical cancer cells (Hela), and lung fibroblasts. By using it for culturing animal cells such as lymphocytes, myeloma cells, leukemia cells, and hybridomas obtained by cell fusion of certain cells with other cells, the cultivation time can be reduced and the amount of cells can be reduced. Increase sales.

Therefore, the animal cell growth promoting oil / fat composition of the present invention can be used without limitation as long as the use requires the growth of animal cells. For example, additives for animal cell growth media, artificial skin agents (cell growth additives), pharmaceuticals, foods, cosmetic ingredients, etc. You can. Hereinafter, these uses will be described.

 [0013] By adding the animal cell growth promoter oil / fat composition of the present invention to an animal cell culture medium, animal cells can be efficiently grown. The animal cell culture medium that can be applied is not particularly limited as long as it is suitable for the animal cell to be grown, and may be a commercially available basic medium or a suitably modified one thereof. . Specific examples of the basic medium include MEM (Minimun Essential Medium) medium, Iskov medium, RPM11640 medium, Ham's F-12 medium, Dulbecco's modified Eagle medium, Dulbecco's modified MEM medium, and Iscov's modified Dulbecco medium with 10% FBS. And preferably Dulbecco's modified Eagle's medium. These media may be used alone or in combination of two or more. The amount of the oil / fat composition of the present invention added to the culture medium may be appropriately adjusted depending on the target animal cell type. In general, the animal cell of the present invention is adjusted so that the concentration in the culture solution is about 0.001 to 100 mg / milliliter, preferably 0.01 to 10 mg / milliliter, and more preferably 0.011 mg / milliliter. A growth promoter oil or fat composition is added.

 When adding the animal cell growth promoter oil / fat composition of the present invention to a medium, it is preferable to add it as a solution. The solvent used for preparing this solution preferably dissolves the animal cell growth promoter oil / fat composition of the present invention and is compatible with the culture solution. Examples of such a solvent include methanol, dimethyl sulfoxide, acetone, and the like, and preferably acetone. These solvents may be used alone or in combination of two or more. From the viewpoint of suppressing the effect of the added solvent on animal cells, the solvent is preferably used so as to be 2% by mass or less based on the total amount of the medium.

It is possible to promote the early formation of artificial skin and increase of artificial skin by adding the animal cell growth promoter oil composition of the present invention to a culture medium for artificial skin production or applying or spraying the composition on cultured artificial skin. it can. Upon application or jetting, a solution or dispersion obtained by dissolving the animal cell growth promoter oil / fat composition of the present invention in an appropriate solvent may be applied or jetted. As the solvent, those similar to the above can be used. In the case of a culture medium for artificial skin production, the solvent is preferably used in an amount of 2% by mass or less based on the total amount of the culture medium as described above. In the case of the solution or dispersion, the solvent can be applied or sprayed within a range that does not adversely affect the cultured human skin. When the animal cell growth promoter oil / fat composition of the present invention is used as a component of pharmaceuticals, foods, and cosmetics, various effects due to promotion of animal cell growth can be expected. A known method can be used for mixing the animal cell growth promoter oil / fat composition of the present invention with a drug or the like.

 [0015] The skin external preparation composition of the present invention may contain other components generally used as skin external preparation components, as long as the effects of the present invention are not impaired. Other components include, for example, purified water, alcohols, oily substances, anti-dandruff agents, bactericides, medicinal ingredients, preservatives, thickeners, astringents, humectants, powders, fragrances, and emulsion stabilizers. And a pH adjuster. When a plant-derived or microorganism-derived glyceride GLA is used, other components derived from a plant or a microorganism may be contained.

 Examples of alcohols include ethanol, higher alcohols having a linear or branched alkyl or alkenyl group, and examples of oily substances include hydrocarbons such as liquid paraffin, vaseline, and solid paraffin; liquid lanolin, lanolin Lanolin derivatives such as fatty acids; silicone derivatives such as dimethylpolysiloxane, polyether-modified polysiloxane, and amino-modified polysiloxane, higher alcohol higher fatty acid esters, higher fatty acids, and fats and oils such as long-chain amidoamine having an alkyl or alkenyl group. Animal and vegetable fats and oils such as mink oil and olive oil; Pharmaceutical ingredients include vitamins and the like. Preservatives include parabens, and thickeners include water-soluble polymer compounds. Examples of the humectant include propylene glycol, glycerin, carbitol, 3-methyl-1,3-butanediol, and saccharides.

[0016] The dosage form of the skin external preparation composition of the present invention is not particularly limited and may be used as it is. Creams, emulsions, lotions, ointments, powders, haptics, powders, and drops , A patch or an aerosol can be applied in the form of a usual external preparation for skin. The amount of the skin external preparation composition of the present invention may be appropriately adjusted depending on the use. For example, when formulated in cosmetics, the concentration in cosmetics can be usually about 0.001 lOOmg / milliliter, preferably 0.01 lOmg / milliliter, more preferably 0.011 lmgZmilliliter. To mix. Also, when used for the treatment of wounds (including pressure sores), the concentration in the therapeutic agent is usually more than 0.001 mgZ milliliter (used as is). The amount is preferably 0.01 to 500 mg / milliliter, more preferably 0.01 to 100 mg / milliliter.

 Example

 Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

 The fatty acid composition of the raw material fats and oils and fat compositions used in the following Examples and Comparative Examples was methyl esterified by the method described in Japanese Patent Publication No. 577-144896 and measured by gas chromatography. The constituent glyceride compositions of the oil and fat compositions of Examples and Comparative Examples were determined based on the Itroscan method (J. Jpn. Oil Chem. Soc., Vol. 35, No. 8, 625-631 (1986)). And Iatroscan TH_10 type (manufactured by Jatron Co., Ltd.) combining a thin layer chromatograph (TLC) and a flame ionization detector (FID). That is, 1 microliter of the sample solution was dropped on one side of a silica gel thin-layer stick (Chromarod S-111, manufactured by Jatron Co., Ltd.), and the solvent was developed. It was attached to type 0 and analyzed under the following conditions.

[0018] Sample solution concentration: 20mg / milliliter acetone

 TLC developing solvent and developing conditions

 Developing solvent: hexane / getyl ether Z acetic acid = 70Z30 / 1 (volume ratio) Developing distance: 10.0cm

 Giatroscan analysis conditions

 Hydrogen flow rate: 160 milliliter / min

 Air flow rate: 2.0 ml Zmin

 Scan speed: 30sec / SCAN

 Detector: Flame ionization detector (FID)

Example 1 (Production of fat composition)

A reaction system containing 100 g of GLA-containing fats and oils having a fatty acid composition shown in Table 1 (manufactured by Idemitsu Kosan Co., Ltd., trade name: Granoy Nore HGC, GLA: 25.9% by mass) and 100 g of distilled water was added to a lipase produced by Rhizopus niveus ( 20,000 units of Newase F3G (manufactured by Amano Enzym Co., Ltd., 30,000 U / g) were added and reacted at 30 ° C. for 48 hours with stirring. [Table 1] Fatty acid composition content (% by mass)

 《Lumic acid 13.5

 Palmitoleic acid 2.6

 Stearic acid 1.7

 Oleic acid 41.9

 Linoleic acid 1 1.7

 r monolinolenic acid 25.9

[0021] After adding 10% by mass aqueous sodium hydroxide solution to the obtained decomposition reaction product to adjust the pH of the decomposition reaction product to 8-9, the oil content in the decomposition reaction product was reduced using 200 ml of getyl ether. After extraction, washing with water and dehydration, ether was removed to obtain 56.4 g of oil (oil / fat composition).

 The glyceride composition analysis was carried out using a part of the oil / fat composition by the above-described method, and the fatty acid composition was analyzed by gas chromatography after a part of the oil / fat composition was methylesterified. The results are shown in Table 3. In Table 3, for example, C16: 0 means a fatty acid having 16 carbon atoms and no double bond, and C18: 3 means a fatty acid having 18 carbon atoms and having 3 double bonds.

Example 2 (Production of Oil and Fat Composition)

 To 100 g of the same GLA-containing fat and oil as used in Example 1, 3 g of water, 30 g of glycerin and 300,000 units of lipase produced by Alcaligenes sp. (Meigyo Sangyo Co., Ltd., Lipase PL, 90, OOOUZg) were added. The mixture was reacted at 30 ° C for 72 hours with stirring.

 A 10% by mass aqueous solution of sodium hydroxide was added to the obtained decomposition reaction product to adjust the pH of the decomposition reaction product to 89, and then oil was extracted from the decomposition reaction product using 200 ml of getyl ether and washed with water. After dehydration, the ether was removed to obtain 87.9 g of an oil component (oil / fat composition).

Using a part of this oil / fat composition, the constituent glyceride composition was analyzed by the method described above, and a part of the oil / fat composition was methylesterified. Was analyzed. The results are shown in Table 3.

 Example 3 (Production of Oil and Fat Composition)

 In Example 1, except that GLA-containing fats and oils having a fatty acid composition shown in Table 2 (manufactured by Idemitsu Kosan Co., Ltd., trade name: Daranoyl CS, GLA: 7.9% by mass) were used in Example 1, An oil / fat composition was obtained by the same enzyme treatment and extraction as in Example 1.

 [Table 2]

Table 3 shows the analysis results of the obtained fat and oil composition.

 Comparative Example 1 (Production of fat composition)

 In Example 1, an oil / fat composition was obtained by performing the same treatment as in Example 1 except that safflower oil containing no GLA acid (manufactured by SIGMA) was used instead of using the GLA-containing oil / fat. Table 3 shows the analysis results of the obtained fat and oil composition.

[Table 3]

Example 1 Example 2 Example 3 Comparative Example 1

 Fatty acid composition (% by mass)

Normitic acid (c _{16: 0} ) 9.2 15.0 16.6 5.4

Palmitoleic acid (c _{16: 1} ) 2.0 2.3 1.9 0.4

Stearic acid (C _{1B: 0} ) 0.0 1.7 2.2 1.8

Oleic acid (c _{18: 1} ) 45.8 40.5 47.9 16.7

Linoleic acid (c _{18: 2} ) 11.7 13.1 17.1 74.9

r Monolinolenic acid (c _{18: 3} ) 29.4 25.7 12.4 0.0

 Glyceride composition (% by mass)

 Triglycerides: TG 59.2 13.8 50.5 62.3

 Diglyceride: DG 37.2 37.9 43.5 33.4

 Monoglyceride: MG 3.6 48.3 6.0 4.3 Example 4 (Cell proliferation test)

 The growth promoting effects of the oil and fat compositions obtained in Examples 13 and 13 and Comparative Example 1 on normal human epidermal keratinocytes were evaluated.

 Normal human epidermal keratinocytes (manufactured by Sanko Junyaku Co., Ltd., Cryo NHEK-Neo: derived from a newborn), bovine pituitary extract included in 500 ml of Bullet Kit KMG basic medium (manufactured by Sanko Junyaku Co., Ltd.) The cells were cultured in a test medium supplemented with 2 ml, 0.5 ml of epidermal growth factor (hEGF) and 0.5 ml of insulin at 37 ° C in the presence of 5% CO for 4 to 7 days.

Those that became 90% subconfluent were used for growth tests.

The proliferation test was performed by seeding normal human epidermal keratinocytes (manufactured by Sanko Junyaku Co., Ltd., Cryo NHEK-Neo: neonatal) at a cell density of 3.5 × 10 ³ cells / well using a 96-well microphone plate. Brett kit KMG basic medium (manufactured by Sanko Junyaku Co., Ltd.) Using a test medium supplemented with 500 ml of bovine brain pituitary extract 2 ml, epidermal growth factor (hEGF) 0.025 ml, and insulin 0.5 ml Incubate for 6 days at 37 ° C, 5% CO

By measuring the number of cells.

The number of cells was measured using a Premix WST-1 Cell Proliferation Assay System (manufactured by Takara Shuzo Co., Ltd.), and the absorbance at 450 nm was measured using a Pell Reader (manufactured by Seikagaku Corporation, model SK601). In addition, the growth rate (%) when the oil / fat composition was added was calculated based on the number of cells in the test medium alone without the oil / fat composition of the present invention as a reference (100%). The results are shown in Table 4. You.

 [0028] [Table 4]

Sample addition amount 0.01 mg / ml () .1 mg / ml

 Sample

 100% without additive

 Example 1 140% 143%

 Example 2 130% 140%

 Example 3 124% 122%

 Comparative Example 1 104% 106%

Example 5 (Cell proliferation and antibody production improvement test)

 The cell growth promoting effects of the oil and fat compositions obtained in Examples 13 and 13 and Comparative Example 1 on CH〇 cells (cultured cells derived from Chinese hamster egg cells) were evaluated. For the growth test, CHO cells (ATCC stock: CRL-11397 strain) were cultured for 7 days at 37 ° C in the presence of 5% CO using CHO-S-SFM II medium (GIBCO BRL) 100 millilitre.

 2

 What had been statically cultured for a while was used.

The proliferation test was performed as follows. That is, the oil and fat compositions obtained in Examples 13 and 13 and Comparative Example 1 were each added to a CHO-S-SFM II medium (manufactured by GIBCO BRL) at a concentration of 0.1 mgZZ to 10 ml of a test medium. 1 × 10 ⁵ cells of CH〇 cells (ATCC storage strain: CRL-11397 strain) were suspended and added to the culture cell space in an integra cell line (CL-350, manufactured by INTEGRA BIOSCIE NCES). In addition, as a basal medium, 200 milliliters of a CHO-S-SFM II medium (GIBCO BRL) without the above-mentioned fat composition was added to the culture cell space.

 The above Integra cell line containing medium and CHO cells was added at 37 ° C in the presence of 5% CO.

After static culture for 2 days, 10 ml of the cell suspension was collected from the cultured cell space. The cell suspension was centrifuged (200 G, 5 minutes), and the cells were placed in a new test medium (CHO-S-SFM II medium (GIBCO BRL)) to obtain the oils and fats obtained in Examples 13 and 13 and Comparative Example 1. Each of the compositions was suspended in a medium supplemented with 0.1 mg / milliliter and added to the cultured cells. Returned to space. After that, static culture was continued again at 37 ° C in the presence of 5% CO for 7 days.

 2

It was.

 After culturing for a total of 14 days, the entire cell suspension was recovered. After the number of cells in this cell suspension was measured using a hemocytometer, the amount of IgG antibody produced in the culture supernatant was measured by quarantine fluorescence (ELISA). In addition, the number of cells and the amount of IgG antibody produced when the same test was performed using only the basal medium (CHO-S-SFM II medium (manufactured by GIBCO BRL)) not containing the oil and fat composition of the present invention were taken as reference (100%). The growth rate (%) and the IgG antibody production improvement rate (%) when the oil and fat composition was added were calculated. Table 5 shows the results.

[Table 5] Sample cell number Improvement rate of IgG antibody production

 No additive 100% 100%

 Example 1 163% 197%

 Example 2 154% 185%

 Example 3 133% 160%

 Comparative Example 1 108% 1 12%

Example 6 (Healing effect test)

 The healing effect when the oil and fat compositions obtained in Examples 13 and 13 and Comparative Example 1 were used as an external preparation for skin was evaluated.

 Glycerin (10 g), liquid paraffin (10 g) and white petrolatum (79 g) were dissolved while heating to 80 ° C, then cooled to 45 ° C, and the oil and fat composition lg obtained in Example 1 was added and mixed. Was quenched to prepare a uniform ointment. This was used as Example 1 ointment. Similarly, an ointment of Example 2, an ointment of Example 3, and an ointment of Comparative Example 1 were prepared using the oil and fat compositions obtained in Examples 2, 3 and Comparative Example 1.

The healing effect of these ointments was evaluated by the following method. In other words, 20 women aged 50 to 60 who have aging symptoms (wrinkles) on the skin are divided into 4 groups of 5 women each, and the first group has Example 1 ointment and the second group has Example 2 ointment. Example 3 ointment for group 3, and ratio of ointment for group 4 Comparative Example 1 The ointment was used for one month, and the condition of wrinkles on the skin before and after use was evaluated according to the following evaluation criteria. Table 6 shows the evaluation results. The numbers in Table 6 indicate the number of people who meet each evaluation criterion.

Evaluation criteria>

 Effective: remarkable disappearance of wrinkles, recovery of epidermal beam.

 Slightly effective: Wrinkles become shallower and less noticeable.

 No effect: No effect before and after use.

[Table 6]

Industrial applicability

 By using the animal cell growth promoter oil / fat composition of the present invention to promote the growth of animal cells, it is possible to improve the productivity of animal cells or the production of useful bioactive substances produced by the cells. it can.

 In addition, the composition for external use on skin of the present invention has an ability to promote epithelial formation in humans and animals, and can be expected to have a healing effect in the treatment of stains and wrinkles, treatment of wounds (including pressure sores), etc. due to metabolism of the epidermis. It is.

Claims

The scope of the claims [1] Triglyceride, shall apply in the fat or oil composition comprising a diglyceride, a monoglyceride and free fatty acids, _I - linolenic acid and _I - Animal cell growth accelerator fat composition containing one or more kinds selected from linolenic acid glyceride.  [2] The animal cell growth promoter according to claim 1, wherein the content of the triglyceride, diglyceride and monoglyceride is 1079% by mass, 20-70% by mass, and 170% by mass, respectively, based on the total amount of glyceride. Fat composition. [3] The animal cell growth promotion according to claim 2, wherein the content of the triglyceride, diglyceride and monoglyceride is 10 to 70% by mass, 25 to 60% by mass and 3 to 65% by mass, respectively, based on the total amount of glyceride. Fats and oils composition. [4] The animal cell growth promoter or fat according to any one of [13] to [13], wherein the content of γ-linolenic acid is 5% by mass or more based on the total amount of the fatty acid and free fatty acid constituting the glyceride. Composition.  [5] The animal cell growth promoter oil / fat composition according to any one of [14] to [14], which is obtained by hydrolyzing or transesterifying a fat or oil in a microorganism or secreted by a microorganism.  6. The animal cell growth promoter oil / fat composition according to claim 5, wherein the microorganism is a bacterium of the genus Mortierella. [7] The animal cell growth promoter / oil composition according to claim 5, wherein the microorganism is a bacterium belonging to the genus Mucor.  [8] A medium for culturing animal cells, comprising the oil / fat composition for animal cell growth promoter according to any one of claims 17 to 17. [9] triglycerides, it shall apply in the fat or oil composition comprising a diglyceride, a monoglyceride and free fatty acids, _I - linolenic acid and _I - skin external composition containing one or more kinds selected from linolenic acid glyceride. [10] The composition for external use on skin according to claim 9, wherein the content of the triglyceride, diglyceride and monoglyceride is 10 to 79% by mass, 20 to 70% by mass and 1 to 70% by mass, respectively, based on the total amount of glyceride. object. 11. The external preparation for skin according to claim 10, wherein the content of the triglyceride, diglyceride and monoglyceride is 10 to 70% by mass, 25 to 60% by mass and 3 to 65% by mass, respectively, based on the total amount of glyceride. object. 12. The composition for external use on skin according to any one of claims 9 to 11, wherein the content of y-linolenic acid is 5% by mass or more based on the total amount of the fatty acid and free fatty acid constituting the glyceride.  13. The external preparation for skin according to any one of claims 9 to 12, which is obtained by a hydrolysis treatment or a transesterification treatment of the fat or oil inside the microorganism or the fat or oil secreted from the microorganism.  14. The skin external preparation composition according to claim 13, wherein the microorganism is a bacterium of the genus Mortierella.  15. The composition for external use on skin according to claim 13, wherein the microorganism is a microorganism belonging to the genus Mucor.