TW200522937A - 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, the present invention relates to a fat composition comprising a triglyceride, a diglyceride, a monoglyceride and a free fatty acid, a fat composition for promoting animal cell growth or an external skin preparation containing at least one member selected from gamma-linolenic acid and gamma-linolenic acid glyceride, and a medium for culturing animal cells containing the fat composition.

Description

200522937 (1) IX. Description of the invention [Technical field to which the invention belongs] The present invention relates to the use of r-linolenic acid (hereinafter referred to as GLA) or r-linolenic acid glyceride which has the ability to promote the proliferation of animal cells. Oil and fat composition, and animal cell culture medium formed from the oil and fat composition. In addition, the present invention relates to a skin external preparation composition containing an oil and fat composition of r-linolenic acid or r-linolenic acid glyceride having an ability to promote cell proliferation of an animal as an effective ingredient. [Prior art] In recent years, it has been shown that with the development of molecular biology, the necessity of animal cell culture has increased significantly. Animal cell culture technology is widely used in cell fusion, manufacturing of single antibodies, and vaccines. It is not only used for research, but also as a cutting-edge technology for the manufacture of disease treatment drugs. The development of biotechnology is also strongly demanded in the society. Animal cell lines cannot proliferate in a medium containing only nutrients such as amino acids, sugars, and salts. In order for animal cells to proliferate, the presence of cell proliferation agents other than simple nutritional components is necessary. Conventionally, as such agents for cell proliferation, various blood pupa such as bovine fetal blood pupa have been used. However, there are many cell proliferation factors in blood pupa, and most of these factors are unknown at this stage, and the content of this factor cannot be determined. Therefore, when the above-mentioned blood pupa is used as a culture medium for animal cells, there is a problem that the cell proliferation activity varies with each culture medium. In addition, the proliferation of hematopoietic cells varies according to the individual animals used, and there is also the problem of instability of hematopoietic cells. On the other hand, in addition to the aforementioned blood pupa, various agents for promoting cell proliferation in animals have been developed. Development of, for example, 5-aminofructoic acid (ami η ο 1 evu 1 inie acid) (for example, refer to Patent Document 1), Rhizopus (Rhizopus) genus-producing substance (RU substance) (for example, refer to Patent Document 2), 5-hydroxyl 2 5 7-dimethoxy-6-methyl-1,4-naphthoquinone (for example, refer to Patent Document 3), carrot seed extract (for example, refer to Patent Document 4), phy to glycogen (For fibroblasts) (for example, refer to Patent Document 5), sugar (monosaccharide, disaccharide), and sugar esters of fatty acids (for example, refer to Patent Document 6). However, these animal cell-promoting agents have problems such as difficulty in production, high production cost, and unstable quality. Therefore, it is required to develop an animal cell proliferation-promoting agent that is inexpensive and excellent in quality stability. In addition, in the field of traditional skin external preparations, many studies have been carried out to activate skin cells, activate the skin function itself, and show improvement of skin symptoms or anti-inflammatory effects or wound healing effects. Traditionally, as external skin-related agents, various substances such as hormonal agents, vitamin agents, r-orytitol, saponin, and other pharmaceutical extracts, lectins, mushroom extracts, and animal proteins have been used. Among these substances, α-hydroxy acid or rosin oil is known to be effective in repairing beryllium lines as a substance for improving skin aging symptoms such as wrinkles or for preventing skin aging or inflammation caused by ultraviolet rays of sunlight. However, because glycolic acid or lactic acid, which are particularly effective among α-hydroxy acids, are oleophilic or lactic acid, etc. -6- 200522937 (3) Lipophilicity, poor percutaneous absorption, and increased side effects such as skin irritation. In addition, rosin oil is oxidized easily in contact with air. Therefore, it is generally used as an ester with fatty acids. However, this fatty acid ester is fat-soluble and it is difficult to mix it with a water-soluble substrate. For the treatment of trauma (surgical incision, gastrointestinal wounds or ulcers, peeling, lacerations, cuts, bedsores, inflammation and surface tissue damage caused by infection), it is believed that it depends on cell proliferation and epithelial tissue formation to stimulate or promote The drugs used in the process of cell differentiation and proliferation included in the wound healing process are effective. An agent that shows healing effects of wounds, also tried to use extracts such as aloe vera as an active ingredient, antibiotics, anti-inflammatory agents, angiotensin, adenine, nicotinic acid, allantoin, vitamin A, zinc, cAMP derivatives (for example, refer to Patent Document 7) and the above-mentioned cell growth factor (b-EGF) (for example, refer to Patent Document 8). However, the above-mentioned effective ingredients sometimes cannot obtain sufficient effects, difficulties in production, high production costs, chemical instability of the effective ingredients, and the like, which require inexpensive and excellent quality stability for skin with an effect of improving skin symptoms or healing effects Topical. Patent Document 1: Japanese Patent Application Laid-Open No. 5 — 49472 Patent Document 2: Japanese Patent Application Laid-Open No. 5 — 9 7 6 8 Japanese Patent Publication 3: Japanese Patent Application Laid-Open No. 5 — 1 3 7 5 6 9 Japanese Patent Publication No. 4: Japanese Patent Application Laid-Open No. 6 — 2 3 9 7 5 9 Patent Document 5: JP 1 1 — 2 5 5 6 5 7 Patent Document 6: JP 2003 — 5 23 66 Patent Document 7: JP Sho 6 3 — 1 0 Patent Publication No. 7 9 3 No. 5 Patent Publication No. 3-1 0 6 8 2 No. 200522937 (4) [Summary of the Invention] Disclosure of the Invention Problems to be Solved by the Invention The present invention has been implemented in view of the above-mentioned facts, The purpose is to provide an inexpensive fat composition with excellent quality stability and the ability to promote animal cell proliferation and an animal cell culture medium containing the fat composition. In addition, the present invention aims to provide inexpensive and stable quality A skin external preparation composition having excellent skin symptom improvement effect or healing effect is intended. Means for Solving the Problem As a result of repeated researches by the present inventors to solve the above-mentioned problems, the inventors discovered that an oil-fat composition containing a specific substance having an ability to promote animal cell proliferation was achieved to achieve the above-mentioned object. The present invention has been completed based on related findings. That is, the present invention provides an oil and fat composition containing triglyceride, diglyceride, monoglyceride, and free fatty acid, and containing one or more agents for promoting animal cell proliferation selected from 7-linolenic acid and r-linolenic acid glyceride. Those who make up oils and fats. Furthermore, the present invention provides an animal cell culture medium containing the oil-fat composition for promoting the proliferation of animal cells. In addition, the present invention provides a fat and oil composition containing triglyceride, diglyceride, monoglycerol-8-200522937 (5) ester and free fatty acid, and one or more selected from r-linolenic acid and r-linolenic glyceride. Skin topical composition. EFFECT OF THE INVENTION According to the present invention, a fat and oil composition for promoting animal cell proliferation, which is inexpensive and excellent in quality stability, and an animal cell culture medium containing the fat and oil composition for promoting animal cell proliferation can be obtained. In addition, a skin external preparation composition having an effect of improving skin symptoms or a healing effect, which is inexpensive and has excellent quality stability, can be obtained. The best form for carrying out the invention is not limited to the fatty acids of triglyceride, diglyceride, and monoglyceride constituting the composition of the animal cell proliferation-promoting agent oil composition or skin external preparation composition of the present invention, and examples can be given. Such as saturated fatty acids and unsaturated fatty acids with 2 to 24 carbon atoms. Examples of suitable fatty acids include caprylic acid, capric acid, myristic acid, myristic acid, pentadecenoic acid, palmitic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, Tetraalkanoic acid, hexacosanoic acid, oleic acid, linoleic acid, α-linolenic acid, r-linolenic acid, octadecanoic acid, eicosinolic acid, eicosinic acid, eicosinic acid Trienoic acid, eicosatetraenoic acid, arachidonic acid, eicosapentaenoic acid, docosenoic acid, docosadienoic acid, docosapentaenoic acid, and docosa carbon Hexenoic acid, etc. There are no particular restrictions on glycerides. Chemical synthetic products, plants, animals, and microorganisms can be used, but it is easy to obtain. 9- 200522937 (6) For plants or microorganisms, should. The content ratio of the triglyceride, diglyceride and monoglyceride contained in the fat composition for animal cell proliferation promoting agent of the present invention or the composition for external skin application is based on the viewpoint that the effect of preventing animal cell proliferation is not uniform. The total amount of glycerides is preferably 10 to 79% by mass, 20 to 70% by mass, and 1 to 70% by mass, respectively. The content ratios of triglyceride, diglyceride, and monoglyceride are more preferably 10 to 70% by mass, 25 to 60% by mass, and 3 to 65% by mass with respect to the total amount of glyceride. The total amount of glycerides is preferably 10 to 65% by mass, 30 to 60% by mass, and 5 to 60% by mass, respectively. The glyceride having such a composition can be produced by a known method. For example, it can be produced by a method of deriving a triglyceride type fat and oil into a diglyceride and a monoglyceride using a chemical catalyst or an enzyme catalyst (for example, refer to Japanese Patent Application Laid-Open No. 2000-2 3 6888 'Japanese Patent Application Laid-Open No. 2000-333688). The GL A series of the active ingredient of the animal cell proliferation-promoting agent oil composition or the skin external preparation composition of the present invention exists not only as a free fatty acid, but also as an ester. The so-called "GLA content" means that all the GLA derivatives in the oil and fat composition of the present invention are all free Gla, free fatty acids obtained by hydrolyzing glycerides, and an oil and fat composition or an external skin composition for an agent for promoting cell proliferation in animals The total amount of free fatty acids is calculated. For example, when GLA is present as an ester, the amount of free GL A obtained by hydrolyzing the ester may be measured. The GLA content in the fat composition for promoting animal cell proliferation can be appropriately adjusted according to the type of animal cells to be promoted. -10- 200522937 (7) Generally, from the viewpoint of preventing the effect of animal cell proliferation due to different types of animal cells, the total amount of fatty acids and free fatty acids constituting glyceride is preferably 5% by mass or more. It is more preferably 10% by mass or more, and more preferably 20% by mass or more. There is no specific upper limit for the content of GLA, but even if it contains more than 98% by mass, the effect of animal cell proliferation will not be further enhanced. From an economic point of view, it is preferably 98% by mass or less. In addition, the GLA content in the composition for external skin preparation may be appropriately adjusted according to the dosage form or type of the composition for external skin preparation. In general, from the viewpoint of preventing a difference in the therapeutic effect due to the dosage form or the use, the total amount of fatty acids and free fatty acids constituting the glyceride is preferably 5% by mass or more. It is more preferably 10% by mass or more, and more preferably 20% by mass or more. There is no specific upper limit for the content of GLA, but even if it exceeds 98% by mass, the effect of animal cell proliferation will not be further enhanced. From an economic point of view, it is preferably 98% by mass or less. GLA is chemically synthesized, and can also be obtained from plant seeds such as borage, evening primrose, and saxifraga, or algae from the genus Spirulina, Cunninghamella, and Mortierella. Microorganisms and genus Mucor. When the obtained GLA is a derivative, it can be used directly, or it can be subjected to hydrolysis treatment or transesterification treatment. The animal cell proliferation-promoting agent oil composition or skin external preparation composition of the present invention can be produced by mixing glyceride and GLA oil and fat at a predetermined ratio as described above by a known mixing method. In addition, the animal cell proliferation-promoting agent oil composition or skin external preparation composition of the present invention is made of GLA-containing microorganisms, and extracts GLA-containing microorganisms' internal oils or microbial secretion oil-11-200522937 (8) GLA grease can be obtained by hydrolysis or transesterification. This GL A-containing oil and fat system will have a microorganism capable of producing GL A-containing oil and fat, which can be obtained by conventional methods. As the microorganism having the ability to produce GLA-containing fats and oils, as described above, various microorganisms such as filamentous fungi, yeast, and algae can be mentioned. For example, as a microorganism having the ability to produce r-linolenic acid-containing fats and oils, for example, the microorganism belonging to the genus Mortierella described in Japanese Patent Application Laid-Open No. 60-1 6 8 3 9 1 and Japanese Patent Application Laid-Open No. 63- Mucor described in 2 83 5 89 and the like belong to the microorganisms, and Rhizopus described in Japanese Patent Laid-Open No. 63-1 3 3 994 and the like belong to the microorganisms. More specifically, as a microorganism belonging to the genus Mortierella, for example, (Mortierella isabellina) IF07824 or (

Mortierella ramaniana var. Angrispora) IF08187 and so on. In addition, as the microorganism belonging to the genus Mucor, for example, (Mucor circi ne 11 〇ides) HUT1121 (FERM P-9 3 5 9) or (Mucor javanicus) HUT1 162 (FERM P-93 60) Wait. The culture medium for culturing the above microorganisms is only required if the above microorganisms have good growth and development, and lipids for the purpose of silicon production. Carbon sources, nitrogen sources, inorganic salts, and amino acids that have good growth and development according to the required microorganisms can be used. By. As the carbon source, sugars such as glucose, starch and waste bran honey, or organic acids or sodium acetate can be used, and sugars such as glucose are particularly preferred. Examples of the nitrogen source include ammonium salts, yeast extracts, corn steep liquor, and peptides. Examples of the inorganic salt include magnesium salt, calcium salt, phosphoric acid salt (-12-200522937 0) salt, iron salt, and copper salt. Regarding culture, when carbon source or nitrogen source or phosphate is added to the culture medium initially, it will cause the microbial growth and development to be adversely affected. Therefore, it can be partially added at an appropriate period after the start of culture to increase the r-linolenic acid. Productivity is better. Other culture conditions', such as temperature and culture time, are determined by considering the composition of the medium or the r-linolenic acid content of the medium or the purpose, and lipid productivity. The temperature is usually in the range of 20 to 3 5 ° c, preferably in the range of 25 to 30 ° c. The pH is usually in the range of 3 to 7, preferably in the range of 3.5 to 6.5, usually in the range of 7 to 2 to 40 hours, to 9 6 to 16 8 hours is appropriate. Since GLA-containing lipids are usually accumulated in microbial cells, the culture solution is solid-liquid separated by a conventional method to obtain GLA-containing cells. According to the use of GLA, although this bacterial cell can also be used directly, in order to further extract and refine fats, the B 1 igh & Dyer method, F ο 1 ich method or JP 5 7 — 1 4 4 9 8 6 The method described in the bulletin and the like can be used for crushing the bacterial cells and extracting the solvent to obtain the intended GLA-containing oil and fat. The GL A-containing oil and fat obtained as described above can be hydrolyzed or transesterified by a known method to obtain an animal cell proliferation-promoting oil or fat composition of the present invention. When the animal cell proliferation-promoting agent oil composition or skin external preparation composition of the present invention is obtained in this way, the GLA content ratio in the animal cell proliferation-promoting agent oil composition or skin external preparation composition is partially promoted. Animal cell proliferation agents, fats and oils, or skin preparations are determined by gas chromatography after methyl esterification (see Japanese Patent Application Laid-Open No. 5 7 — 1 449 86). 200522937 (10) The fat or oil composition for promoting animal cell proliferation of the present invention may contain other ingredients as long as it does not inhibit the effects of the present invention. Examples of the other component 'include the above fatty acids other than GLA. When using plant-derived or microorganism-derived glycerides or GLA, it may contain other ingredients derived from plants and microorganisms. The animal cell proliferation promoting agent oil composition of the present invention is based on the use of cultured epidermal keratinocytes, skin fibroblasts, melanocytes, small intestinal epithelial cells, human cervical cancer cells (Hela), and lung fibroblasts. Animal cells such as anchor dependent cells 11, lymphocytes, bone marrow tumor cells, leukemia cells, and hybridoma cells obtained by the fusion of a cell with other cells can shorten the culture time With increased cell volume. However, the oil-fat composition of the agent for promoting animal cell proliferation of the present invention can be used without limitation as long as the application for animal cell proliferation is required. Examples include additives for animal cell proliferation media, artificial skin agents (cell proliferation additives), pharmaceuticals, food, and cosmetic ingredients. The following is a description of these uses. By adding the fat composition for promoting animal cell proliferation of the present invention to an animal cell culture medium, animal cells can be efficiently proliferated. The applicable medium for animal cell culture is not particularly limited as long as it is suitable for proliferating animal cells, and it may be a commercially available basic medium or may be appropriately modified. As specific basic medium, for example, MEM (Minimnn Essential Medium) medium, Iscove's medium, RPM 1 1 640 medium, Ham, s f-12 medium, Diilbecco, s 200522937 (11) modified Eagle's medium (DMEM) medium, Dulbecco's modified MEM Medium and IMDM medium (Iscove's Modified Dulbecco's Medium) with 10% FBS, DMEM medium is suitable. These medium lines can be used alone or in combination of two or more. The amount of the fat composition of the present invention added to the culture medium depends on the type of the target animal cell. It can be adjusted appropriately. Generally, the fat composition of the animal cell proliferation promoting agent of the present invention is added so that the concentration in the culture solution becomes 0.001 to 100 mg The degree of / liter is preferably from 0.001 to 10mg / liter, and more preferably from 0.001 to img / liter_. When the fat composition of the animal cell proliferation agent of the present invention is added to the culture medium It is preferable to add it as a solution. The solvent used to prepare this solution is to dissolve the fat composition of the animal cell proliferation promoting agent of the present invention, and it is preferably soluble in the culture liquid phase. As such a solvent, for example, methanol, Acetone is suitable for dimethyl asus, acetone, etc. These solvents can be used alone or in combination of two or more. In addition, from the viewpoint of suppressing the effect of the added solvent on animal cells, the use of the solvent system for the total medium It is preferable that the content is 2% or less. Add the fat composition for promoting animal cell proliferation of the present invention to the culture medium for artificial skin production, or apply or spray on the culture medium. 'Artificial skin' can promote the early formation of artificial skin or increase in artificial skin. When applying or spraying, the animal cell proliferation promoting agent oil composition of the present invention can also be dissolved in a suitable solvent solution or dispersed dispersion coating or Spray. As the solvent system, the same ones as described above can be used. When the culture medium for artificial skin production is used, the solvent system is used in the same manner as above, using 2% by mass of the total culture medium. -15-200522937 (12) or less. In addition, In the above solution or dispersion, the solvent can be applied or sprayed within a range that does not cause adverse effects on the cultured artificial skin. The oil and fat composition using the animal cell proliferation promoting agent of the present invention is a medical pleasure product, food or cosmetic ingredient In this case, various effects of promoting the proliferation of animal cells can be expected. Known methods can be used for mixing the animal cell proliferation-promoting agent oil and fat composition of the present invention with medicinal products and the like. In the skin external preparation composition of the present invention, Those who do not inhibit the effects of the present invention may contain other ingredients generally used as skin external preparations. As other ingredients Examples include refined water, alcohols, oily substances, anti-dandruff agents, bactericides, medicinal ingredients, preservatives, thickeners, astringents, humectants, powders, perfumes, emulsifier stabilizers and pH adjusters. Etc. When using plant-derived or microorganism-derived glycerides or GLA, it may also contain other ingredients derived from plants or microorganisms. Examples of the alcohols include ethanol, linear or branched alkyl or alkenyl groups. Higher alcohols include oily substances such as liquid paraffins, petroleum jelly, and solid paraffins; lanolin derivatives such as liquid lanolin and lanolin fatty acids; dimethyl polysiloxanes, Polyether modified polysiloxanes and amino modified polysiloxanes, such as polysiloxane derivatives, higher alcohols, higher fatty acid esters, higher fatty acids, long-chain ammonium amines with alkyl or alkenyl oils and fats; Animal and vegetable oils such as mink oil and olive oil. Examples of the medicinal ingredient 'include vitamins. Examples of the preservative include parabens, and examples of the thickener include water-soluble polymer compounds. Examples of the humectant include propylene glycol, glycerin, carbitol, 3-methyl-1,3-butanediol, and sugars. -16- 200522937 (13) There are no particular restrictions on the dosage form of the skin external preparation composition of the present invention, and it can be used directly, and it can also be applied in cream, emulsion, lotion, ointment, powder, paste Formulations, powders, drops, patches, aerosols, etc. are usually external skin preparations.

The amount of the skin external preparation composition of the present invention can be appropriately adjusted depending on the application. For example, when blending cosmetics, the concentration in the cosmetics can usually be about 0.001 to 100 mg / liter, preferably 0.01 to 10 mg / liter, and 0.01 to 1 mg / liter. In addition, when used in the treatment of trauma (including bedsores), the concentration in the therapeutic drug can usually be 0.001 mg / liter or more ~ (direct use), it is preferably 0.01 to 500 mg / liter, and 0.01 to lOOmg / liter is particularly preferred. [Embodiments] Examples Next, the present invention will be described in more detail with examples, but the present invention is not limited to those examples. The fatty acid composition of the raw fats and oils and fats and oils composition using the following examples and comparative examples was methyl esterified by the method described in Japanese Patent Application Laid-Open No. 5 7-1 448 96 and measured by gas chromatography. The glyceride composition of the oil and fat compositions of Examples and Comparative Examples is based on the Iatroscan method (J. Jpn. Oil Chem. Soc ·, V ο 1 3 5? N ο 8 5 625-631 (1986)), It is obtained by combining thin layer chromatography (TLC) and hydrogen flame ion detector (FID) of Iatroscan TH_10 (manufactured by Iatros). That is, one microliter of the sample solution was dropped on one side of a silicon thin-layer rod (Chr 0ma 100d S II, I atr 0s -17- 200522937 (14)), and the solvent was developed. After the solvent was unfolded, a thin silicon rod was mounted on the above-mentioned analyzer TH-10 and analyzed under the following conditions. Sample solution concentration: 20 mg / ml acetone TLC development solvent: hexane / diethyl ether / acetic acid = 70/3 0/1 (volume ratio) development distance: 10.0cm I atr 〇sca η analysis conditions hydrogen flow rate: 160 Liters / min Air flow: 2.0 liters / min Scanning speed: 30sec / SCAN Detector: Hydrogen Flame Ion Detector (FID) Example 1 (Production of grease composition)

A reaction system containing 100 g of GLA-containing oils and fats with the fatty acid composition shown in Table 1 (made by Idemitsu Kosan Co., Ltd., trade name: GlanoilHGC, GLA: 25.9% by mass) and 100 g of distilled water, adding 20,000 units Rhizopus niveus produces lipase (made by Amano Enzyme (stock),

Newlase F3G, 3 0,000 U / g), and stirred at 30 ° C for 4 8 hours. -18- 200522937 (15) [Table 1] Fatty acid composition Content rate (% by mass) Palmitic acid 13.5 Palmenic acid 2.6 Stearic acid 1.7 Oleic acid 4 1.9 Linoleic acid 11.7 r-Linolenic acid 25.9

10 mass% sodium hydroxide aqueous solution was added to the obtained decomposition reaction product, and after adjusting the pH of the decomposition reaction product to 8 to 9, the oil content in the decomposition reaction product was extracted with 200 ml of diethyl ether, washed with water, and dehydrated, and then removed Ether to obtain 56.4 g of oil content (fat composition). A part of this oil and fat composition was analyzed by the above-mentioned method to constitute glyceride. In addition, the methyl esterified part of the oil and fat composition was analyzed by gas chromatography. The results are shown in Table 3. In addition, in Table 3, for example, C1 6: 0 means a fatty acid having 16 carbon atoms and no double bond, and C1 8: 3 means a fatty acid having 18 carbon atoms and 3 double bonds. Example 2 (Production of fat and oil composition) To 100 g of the same GLA-containing fat as the user of Example 1, 3 g of water, 30 g of glycerol, and 30,000 units of Alcaligenes sp · produced lipase (named Sugar industry (stock), LipasePL, 90,000U / g -19- 200522937 (16)), under stirring, react at 30 ° C for 7 2 hours. Add a 10% by mass sodium hydroxide aqueous solution to the obtained decomposition reaction product, adjust the pH of the decomposition reaction product to 8 to 9, and use 200 ml of diethyl ether to extract the oil content in the decomposition reaction product, wash with water, and dehydrate, and then remove Ether to obtain 8 7.9 g of oil content (fat composition). A part of this oil and fat composition 'was used to analyze glyceride composition by the method described above, and the methyl esterified part of the oil and fat composition was analyzed by gas chromatography. The results are shown in Table 3. Example 3 (Production of oil and fat composition) In Example 1, except that a GLA-containing oil and fat having a fatty acid composition as shown in Table 2 (manufactured by Idemitsu Kosan Co., Ltd., trade name: GlanoilCS, GLA: 7.9% by mass) was used Except for GLA fats and oils, a fats and oils composition was obtained by the same enzyme treatment and extraction as in Example 1. [Table 2] Fatty acid composition content ratio (mass%) Palmitic acid 3 1.7 Palmenoic acid 1.8 Stearic acid 2.6 Oleic acid 40.7 Linoleic acid 14.2-Linolenic acid 7.9 -20- 200522937 (17) Fat and oil composition obtained The analysis results are shown in Table 3. Comparative Example 1 (Production of fat and oil composition) In Example 1, a safflower oil (manufactured by SIGMA) without GLA acid was used instead of using GLA-containing fat and oil, and the same treatment as in Example 1 was performed to obtain a fat and oil composition. . Table 3 shows the analysis results of the obtained fat and oil composition.

[Table 3] Example 1 Example 2 Example 3 1 Comparative Example 1 Fatty acid composition (mass%) palmitic acid (C 1 6: 〇) 9.2 15.0 16.6 5.4 palmitic acid (C 1 6: 1) 2.0 2.3 1. 9 0.4 stearic acid (c 1 8: 〇) 0.0 1. .7 2.2 1.8 oleic acid (c 1 8: 1) 45.8 40.5 47.9 16.7 linoleic acid (C) 8 ·· 2) 11.7 13.1 17.1 74.9 τ-linolenic acid (C18: 3) 29.4 25.7 12.4 0.0 Glyceride composition (% by mass) Triglyceride: TG 59.2 13.8 50.5 52.3 Diglyceride: DG 37.2 37.9 43.5 33.4 Monoglyceride · MG 3.6 46.3 6.0 4.3 Example 4 (Cell proliferation Test) The effects of the oil and fat compositions obtained in Examples 1 to 3 and Comparative Example 1 on the proliferation promotion of -21-200522937 (18) in normal human epidermal keratinocytes were evaluated.

Normal human epidermal keratinocytes (manufactured by Sanko Pure Chemicals Co., Ltd., Cryo NHEK-Neo: from neonates) were used in 500 ml of BlettkitKMG basic medium (manufactured by Sanko Pure Chemicals Co., Ltd.), and 2 ml Auxiliary bovine pituitary extract, 0.5 liters of epithelial growth factor (hEGF) and 0.5 liters of insulin test medium, cultured at 37 ° C, 5% C02 for 4 to 7 days, use Become 70 to 90% subconfluent in the proliferation test. The proliferation test was performed using a 96-well micro-culture plate at a cell density of 3.5 X 103 cells / well, and inoculated with normal human epidermal keratinocytes (manufactured by Sanko Pure Pharmaceutical Co., Ltd., Cryo NHEK—Neo: from newborns), used in 5,000 ml of BlettkitKMG basic medium (made by Sanko Pure Pharmaceutical Co., Ltd.), supplemented with 2 liters of attached bovine pituitary extract, 0.025 liters of epithelial growth factor (hEGF), and 0.5 liters of insulin as test medium Incubate at 37 ° C and 5% C02 for 6 days. The cell number was measured using the Premix WST-1 Cell Proliferation Assay System (made by Takara Shuzo Co., Ltd.), and the well leader (Biochemical Industry (Stock)) System, SK 60 01 type) was measured at 450 nm absorption. In addition, based on the number of cells in the test medium containing no fat or oil composition of the present invention (100%), the proliferation rate (%) when the fat or oil composition was added was calculated. The results are shown in Table 4. -22- 200522937 (19) [Table 4] Sample addition amount 0.01 mg / ml 0.1 mg / ml Sample non-added 100% Example 1 140% 143% Example 2 13 0% 140% Example 3 124% 122% Comparison Example 1 104% 106%

Example 5 (Test of Cell Proliferation and Promoting Antibody Production) The effects of the oil and fat compositions obtained in Examples 1 to 3 and Comparative Example 1 on the promotion of CHO cells (cultured cells from Chinese hamster egg cells) were evaluated. Using 100 liters of CHO-S-SFMII medium (manufactured by GIBCOBRL), CHO cells (ATCC preservation strain: CRL-1 1 3 97 strain) were cultured at 37 ° C in the presence of 5% C02 for 7 hours. The celestial being used in the proliferation test. The proliferation test was performed as follows. That is, the oil and fat composition obtained in each of Examples 1 to 3 and Comparative Example 1 was added to a 10-liter test medium of CHO-S-SFMII medium (manufactured by GIBCOBRL) at a concentration of 0.1 mg / 亳 liter, and suspended for 1 minute. X 1 05cell CHO cells (ATCC preservation strain: CRL-1 1 3 97 strain) were added to the cultured cell space in INTEGRA CELLINE (made by INTEGRA BIOSCIENCES, CL-3 50). In addition, 200 liters of CHO-S-SFM Π medium (manufactured by GIBCOBRL) without the above-mentioned fat composition was used as a basic medium, and added to the culture cell space -23-200522937 (20). 10N of the above-mentioned 1NTEGRA CELLINE supplemented with culture medium and CH0 cells was allowed to stand for 7 days in the presence of 37 ° C, 5% CO2, and then 10 l of a cell suspension was recovered from the cell space of the culture solution. This cell suspension was centrifuged (200 G, 5 minutes). The cells were suspended in a new test medium (at a concentration of 0.1 mg / liter, each of Examples 1 to 3 and Comparative Example 1 were added. The obtained oil-and-fat composition was placed in a CHO-S-SFM Π culture medium (manufactured by GIBCOBRL), and this was returned to the cultured cell space. Thereafter, the cells were allowed to stand for 7 days at 37 ° C in the presence of 5% C02. The total amount of cell suspension was recovered after 14 days of culture. After measuring the number of cells in this cell suspension using a hemocytometer, the amount of IgG antibodies produced in the culture supernatant was measured by enzyme-linked immunosorbent assay (ELISA method). In addition, based on the number of cells and the amount of IgG antibody produced when the same test was performed on a basic medium (CHO-S-SFM Π medium (manufactured by GIBCOBRL)) containing only the oil and fat composition of the present invention, it was calculated as a reference (100%). When the fat composition is added, the proliferation rate (%) and the IgG antibody production rate (%) are increased. The results are shown in Table 5. -24-200522937 (21) [Table 5] Increase in the number of sample cells No production of IgG antibodies 10 0% 1 0 0% Example 1 1 6 3% 1 9 7% Example 2 1 5 4% 18 5% Implementation Example 3 1 3 3% 1 6 0% Comparative Example 1 108% 1 12% Example 6 (Healing Effect Test) Evaluation of the oil and fat composition obtained using Examples 1 to 3 and Comparative Example 1 as a composition for external skin application The healing effect of time. 10 g of glycerin, 10 g of flowing paraffin and 79 g of white vaseline were heated to 80 ° C and dissolved, and then cooled to 4 5 ° C, and 1 g of the oil and fat composition obtained in Example 1 was added and mixed. This mixture is rapidly cooled to prepare a uniform ointment. This was used as Example 1 ointment. Similarly, the fat and oil compositions obtained in Examples 2 and 3 and Comparative Example 1 were used to prepare the ointment of Example 2 and the ointment of Example 3 and the ointment of Comparative Example 1. The healing effects of these ointments were evaluated by the following methods. In other words, 20 women aged 50 to 60 with skin aging symptoms (streaking lines) were divided into 4 groups of '5 in each group'. The first group was Example 丨 ointment, and the second group was Example 2 Ointment 'The third group is Example 3 ointment and the fourth group is Comparative Example 1 Ooxiao' 1 month of use 'The skin wrinkle condition before and after use was evaluated according to the following evaluation criteria. The evaluation results are shown in Table 6. In addition, the numbers in Table 6 are the number of people who meet the respective evaluation criteria. -25- 200522937 (22) <Evaluation Criteria> Effective ... It shows that wrinkles have disappeared obviously and the epidermal tension is restored. Slightly effective: It shows the improvement effect of wrinkles becoming lighter and less noticeable. Invalid: no effect before and after use. [Table 6] Evaluation Example 1 Example 2 Example 3 Comparative Example 1 Ointment Ointment Ointment Ointment Effective 3 4 1 0 Slightly effective 2 1 3 1 Ineffective 0 0 1 4

Industrial Applicability By using the oil-fat composition for promoting animal cell proliferation of the present invention to promote animal cell proliferation, it is possible to improve the productivity of animal cells, or to improve the productivity of effectively producing cell physiologically active substances. In addition, the skin external preparation composition of the present invention has the ability to promote the formation of human and animal epithelium, and can be expected to cure the dark spots caused by the epidermal metabolism, countermeasures against wrinkles, and the treatment of wounds (including bedsores). -26-

Claims (1)

200522937 (1) 10. Scope of patent application1. An oil and fat composition for promoting animal cell proliferation, characterized in that it contains a triglyceride, a diglyceride, a monoglyceride and a free fatty acid, containing more than one selected From r-linolenic acid and r-linolenic glyceride. 2. The fat composition for an animal cell proliferation promoting agent according to item 1 of the application, wherein the content ratio of the triglyceride, diglyceride and monoglyceride is 10 to 79 masses for the total amount of glycerides. %, 20 to 70% by mass, and 1 to 70% by mass. 3. The fat composition for an animal cell proliferation promoting agent according to item 2 of the patent application, wherein the content ratios of the triglyceride, diglyceride and monoglyceride are 10 to 70 masses for the total amount of glycerides. %, 25 to 60% by mass, and 3 to 65% by mass. 4. The fat and oil composition for an animal cell proliferation agent according to any one of claims 1 to 3, wherein the content ratio of r-linolenic acid is the total amount of fatty acids and free fatty acids constituting the glyceride It is 5 mass% or more. 5. The oil and fat composition for promoting animal cell proliferation according to any one of the claims 1 to 4 of the scope of application for a patent, wherein the oil or fat is produced by hydrolysis treatment or transesterification of microbial body oil or microbial secretion oil. 6. An oil and fat composition for an animal cell proliferation promoting agent, such as in item 5 of the scope of the patent application, wherein the microorganism is a bacterium of the genus Mortierella. 27- 200522937 (2) An oil and fat composition, wherein the microorganism is a Mucor genus. 8. A culture medium for animal cell culture, characterized in that it contains an oil and fat composition for promoting animal cell proliferation as described in any one of claims 1 to 7 of the scope of patent application. 9. A skin external preparation composition, characterized in that it comprises a triglyceride, a diglyceride, a monoglyceride and a free fatty acid oil composition containing at least one selected from r-linolenic acid and r-linolenic glyceride. ίο. According to the skin external preparation composition according to item 9 of the application, wherein the content ratios of the triglyceride, diglyceride and monoglyceride are 10 to 79% by mass and 20 to 70% of the total amount of glycerides, respectively. Mass% and 1 to 70 mass%. 11. The external skin composition according to item 10 of the application, wherein the content ratios of the triglyceride, diglyceride and monoglyceride are 10 to 70% by mass of the total glyceride, 25 to 60% by mass and 3 to 65% by mass. 12. The skin external preparation composition according to any one of claims 9 to 11 in the scope of the patent application, wherein the content ratio of 7-linolenic acid is 5 for the total amount of fatty acids and free fatty acids constituting the glyceride % By mass or more 013. The skin external preparation composition according to any one of claims 9 to 12 of the scope of application for a patent, which is obtained by hydrolysis treatment or transesterification treatment of microbial fats or oils secreted by microorganisms. 14. The skin external preparation composition according to item 3 of the scope of application for a patent, wherein the microorganism is a genus of Mortierella. -28- 200522937 (3) 1 5. The skin external preparation composition according to item 13 of the patent application scope, wherein the microorganism is a genus of Mucor. -29- 200522937 Qiming said that the single abbreviation number is the symbol designation table of the symbolic table: the plan and the table, the table, and the table. Reveal the chemical formula that best characterizes the invention: None