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) 九、發明說明 【發明所屬之技術領域】 本發明係關於含具有促進動物細胞增殖能力之r -亞 麻酸（以下簡稱爲GLA )或r -亞麻酸甘油酯之促進動物 細胞增殖用劑油脂組成物，及由此油脂組成物所形成之動 物細胞培養用培養基。 另外，本發明係關於含具有促進動物細胞增殖能力之 r -亞麻酸或r -亞麻酸甘油酯之油脂組成物爲有效成份 之皮膚外用劑組成物。 【先前技術】 近年來，顯示隨著分子生物學發展，動物細胞培養之 必要性明顯地增加。動物細胞培養技術係廣泛地使用於細 胞融合、單株抗體之製造及疫苗之製造等，不僅於硏究， 作爲疾病治療藥製造之先端技術，社會上亦強烈地要求該 發展之生物技術。 動物細胞係無法於僅含胺基酸、糖分及鹽類等營養成 份之培養基中增殖，爲使動物細胞增殖，單純的營養成份 以外之細胞增殖用劑之存在係必要的。 傳統上，作爲如此細胞增殖用劑，使用牛胎兒血淸等 各種血淸。然而，血淸中存在多數個細胞增殖因子，而且 現階段該因子多爲未知成份，亦無法測定該因子含量。因 此，使用上述血淸爲動物細胞用培養基時，隨著各個培養 基而有細胞增殖活性不齊之問題。另外，血淸細胞增殖因 -5- 200522937 (2) 子活性係依所採取動物個體差異而有變動，亦有血淸品霄 不穩定之問題。 另一方面，除了上述血淸以外，亦開發各種促進動物 細胞增殖用劑。開發例如5 —胺基果糖酸（a m i η ο 1 e v u 1 i n i e acid )(例如參考專利文獻1 )、根黴（Rhizopus)屬產生物 質（RU物質） （例如參考專利文獻2 ) 、5 -羥基一 2 5 7 一二甲氧基一 6 —甲基一 1，4 一萘醌（例如參考專利文獻3 )、御種紅蘿蔔萃取液（例如參考專利文獻4 )、植物肝 醣（phy to glycogen)(纖維母細胞用）（例如參考專利文獻 5 )、糖（單糖、雙糖）及脂肪酸之糖酯（例如參考專利 文獻6 )等之促進動物細胞用劑。然而，此等之促進動物 細胞用劑係有生產困難、生產成本高及品質不安定等問題 ，要求開發價廉且品質安定性優異之促進動物細胞增殖用 劑。 另外，傳統之皮膚外用劑領域中，實施許多賦活化皮 膚細胞，活化皮膚機能本身，顯示改善皮膚症狀或抗炎症 效果或創傷治癒效果之硏究。傳統上作爲相關的皮膚外用 劑，使用激素劑、維生素劑、r -谷維醇及皂角苷等之生 藥萃取物、植物凝集素、菇類萃取物，進而來自動物之蛋 白質等之各種物質。 此等物質中，作爲具有改善皺紋等之皮膚老化症狀作 用之物質或防止因陽光紫外線所引起皮膚老化或炎症作用 之物質，已知α -羥酸或松香油有效地地修復鈹紋。然而 ，因爲α -羥酸中顯示尤其有效作用之乙醇酸或乳酸等係 -6- 200522937 (3) 親油性，經皮吸收差，增加配合量時，顯示皮膚刺激等之 不良副作用。另外，因松香油接觸空氣容易氧化，所以通 常作爲與脂肪酸之酯體使用，但此脂肪酸酯係脂溶性，所 以難以配合於水溶性基材。 於治療創傷（外科之切開、腸胃管的傷口或潰瘍、剝 離、裂傷、切斷、褥瘡、發炎及感染所引起的表面組織損 傷）上，認爲依賴細胞增殖及上皮組織形成，刺激或促進 於創傷治癒過程所包含之細胞分化、增殖過程之藥劑係有 效的。顯示創傷治癒效果之藥劑，亦嘗試使用作爲有效成 份之蘆薈等萃取物、抗生素、抗炎症劑、血管舒緩素、腺 嘌呤、煙酸、尿囊素、維生素A、鋅、cAMP衍生物（例 如參考專利文獻7 )，以及上述之細胞成長因子（b - EGF )(例如參考專利文獻8 )。然而，上述有效成份有時不 能得到充份的效果，生產困難，生產成本高，有效成份化 學上不安定等之問題，要求價廉且品質安定性優異之具有 改善皮膚症狀效果或治癒效果之皮膚外用劑。 專利文獻1 :特開平5 — 49472號公報 專利文獻2 :特開平5 — 9 7 6 8 5號公報 專利文獻3 :特開平5 — 1 3 7 5 6 9號公報 專利文獻4 :特開平6 — 2 3 9 7 5 9號公報 專利文獻5 :特開平1 1 — 2 5 5 6 5 7號公報 專利文獻6 :特開2003 — 5 23 66號公報 專利文獻7 :特開昭6 3 — 1 0 7 9 3 5號公報 專利文獻8 :特開平3 - 1 0 6 8 2 3號公報 200522937 (4) 【發明內容】 發明之揭示 發明所欲解決之課題 本發明係有鑑於上述事實而實施者，係以提供價廉且 品質安定性優異，具有促進動物細胞增殖能力之油脂組成 物及含有此油脂組成物之動物細胞培養用培養基爲目的者 〇 另外，本發明係以提供價廉且品質安定性優異之具有 改善皮膚症狀效果或治癒效果之皮膚外用劑組成物爲目的 者。 課題之解決手段 本發明者等係爲解決上述課題而重覆努力硏究的結果 係發現由含有具有促進動物細胞增殖能力之特定物質之油 脂組成物，而達成上述目的。本發明係基於相關發現而完 成者。 亦即，本發明係提供含三甘油酯、二甘油酯、單甘油 酯及游離脂肪酸之油脂組成物，含有一種以上選自7 -亞 麻酸及r -亞麻酸甘油酯之促進動物細胞增殖用劑油脂組 成物者。 並且’本發明係提供含有此促進動物細胞增殖用劑油 脂組成物之動物細胞培養用培養基者。 另外，本發明係提供含三甘油酯、二甘油酯、單甘油 -8 - 200522937 (5) 酯及游離脂肪酸之油脂組成物，含有一種以上選自r -亞 麻酸及r -亞麻酸甘油酯之皮膚外用劑組成物者。 發明之功效 依據本發明而可得到價廉且品質安定性優異之促進動 物細胞增殖用劑油脂組成物，及含有此促進動物細胞增殖 用劑油脂組成物之動物細胞培養用培養基。另外，可得到 價廉且品質安定性優異之具有改善皮膚症狀效果或治癒效 果之皮膚外用劑組成物。 用以實施發明之最佳型態 對於構成本發明之促進動物細胞增殖用劑油脂組成物 或皮膚外用劑組成物成份之三甘油酯、二甘油酯及單甘油 酯之脂肪酸並無限制，可舉例如碳原子數爲2至24個之 飽和脂肪酸及不飽和脂肪酸。作爲適合之脂肪酸，可舉例 如辛酸、癸酸、肉豆蔻酸、肉豆蔻烯酸、十五碳烯酸、棕 櫚酸、棕櫚烯酸、硬脂酸、花生酸、二十二烷酸、二十四 烷酸、二十六烷酸、油酸、亞油酸、α —亞麻酸、r 一亞 麻酸、十八院四條酸、二十碳丨希酸、二十碳二燃酸、二十 碳三烯酸、二十碳四烯酸、花生浸烯酸、二十碳五烯酸、 二十二碳烯酸、二十二碳二烯酸、二十二碳五烯酸及二十 二碳六烯酸等。 作爲甘油酯，並無特別的限制，化學合成品、來自植 物、動物者、來自微生物者皆可使用，但就容易取得之觀 -9- 200522937 (6) 點上，以來自植物或微生物者爲宜。 本發明之促進動物細胞增殖用劑油脂組成物或皮膚外 用劑組成物中所含之三甘油酯、二甘油酯及單甘油酯之含 有比率’就爲防止動物細胞增殖效果不齊之觀點，係對於 甘油酯總量，以分別爲1 0至7 9質量％、2 0至7 0質量％ 、1至70質量％爲宜。三甘油酯、二甘油酯及單甘油酯之 含有比率，係對於甘油酯總量，以分別爲1 〇至70質量％ 、25至60質量％、3至65質量％尤佳。對於甘油酯總量 ，以分別爲10至65質量％、30至60質量％、5至60質 量％最好。 如此組成之甘油酯係可以已知方法而可製造。例如由 將三甘油酯型油脂，使用化學催化劑或酵素催化劑，衍生 成二甘油酯及單甘油酯之方法（例如參考特開2000 -2 3 6888’特開2000— 333688號公報）而可製造。 本發明之促進動物細胞增殖用劑油脂組成物或皮膚外 用劑組成物之有效成份之G L A係不僅作爲游離脂肪酸， 亦可作爲酯而存在。所謂「GLA含量」係指以本發明之油 脂組成物中之GLA衍生物全部爲游離Gla，由水解甘油 酯所得之游離脂肪酸以及存在於促進動物細胞增殖用劑油 脂組成物或皮膚外用劑組成物之游離脂肪酸之合計所算出 。例如GLA作爲酯存在時，測定水解此酯所得之游離 G L A量即可。 促進動物細胞增殖用劑油脂組成物中之GLA含量係 因應成爲促進增殖對象之動物細胞種類而可適當地調整。 -10- 200522937 (7) 一般就防止因動物細胞種類相異而增殖動物細胞效果不齊 之觀點上，對於構成甘油酯之脂肪酸及游離脂肪酸之合計 量，以5質量％以上爲宜。以1 0質量％以上尤佳，以20 質量％以上更好。GLA含量並無特定的上限，但即使含超 過9 8質量％，並不會更提昇動物細胞增殖效果，就經濟 觀點上，以98質量％以下爲宜。 另外，皮膚外用劑組成物中之GLA含量係因應皮膚 外用劑組成物之劑型或種類，適當地調整即可。一般就防 止因劑型或使用用途而治療效果不齊之觀點上，對於構成 甘油酯之脂肪酸及游離脂肪酸之合計量，以5質量％以上 爲宜。以1 〇質量％以上尤佳，以20質量％以上更好。 GLA含量並無特定的上限，但即使含超過98質量％，並 不會更提昇動物細胞增殖效果，就經濟觀點上，以9 8質 量％以下爲宜。 GLA係可化學合成而得，亦可得自琉璃苣、月見草及 虎耳草等之植物種子或螺旋藻（Spirulina )屬等之藻類、 小克銀漢黴（Cunninghamella)屬、被孢黴（Mortierella )屬及毛黴（Mucor )屬等之微生物。所得之GLA爲衍生 物時，可直接使用，亦可進行水解處理或酯交換處理。 本發明之促進動物細胞增殖用劑油脂組成物或皮膚外 用劑組成物係將甘油酯與GLA油脂以上述所定比率，以 已知混合方法混合而可製造。另外，本發明之促進動物細 胞增殖用劑油脂組成物或皮膚外用劑組成物係由含GLA 之微生物，萃取含GLA微生物體內油脂或微生物分泌油 -11 - 200522937 (8) 月旨，將此含GLA油脂，由水解處理或酯交換處理而可得 之。 此含G L A油脂係將具有生產含G L A油脂#力之微生 物，由常法培養而可得之。作爲具有生產含GLA油脂能 力之微生物，如上所述，可舉例如絲狀菌或酵母、藻類等 之各種者。例如，作爲具有生產含r -亞麻酸油脂能力之 微生物，可舉例如特開昭60 - 1 6 8 3 9 1號公報等所記載之 被孢黴（Mortierella)屬所屬微生物、特開昭63 - 2 83 5 89 號公報等所記載之毛黴（Mucor )屬所屬微生物、特開昭 63 — 1 3 3 994號公報等所記載之根黴（Rhizopus )屬所屬微 生物等。 更具體地，作爲被孢黴（Mortierella)屬所屬微生物 ，可舉例如（Mortierella isabellina ) IF07824 或（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 等。另夕t ，作爲毛黴（Mucor )屬所屬微生物，可舉例如（Mucor c i r c i ne 11 〇 i d e s ) HUT1121 ( FERM P - 9 3 5 9 )或（M u c o r javanicus ) HUT1 162 ( FERM P-93 60 )等。 培養上述微生物用之培養基係只要上述微生物生長發 育良好，矽生產目的之脂質者即可，可使用含碳來源、氮 來源及無機鹽類及因應所需之微生物生長發育良好的胺基 酸等成份者。作爲碳來源，可使用葡萄糖、澱粉及廢糠蜜 等糖類或有機酸或醋酸鈉等，以葡萄糖等之糖類尤佳。另 外，作爲氮來源，可舉例如銨鹽、酵母萃取物、玉米浸漬 液及胜肽等。作爲無機鹽類，可舉例如鎂鹽、鈣鹽、磷酸 -12- 200522937 Ο) 鹽、鐵鹽及銅鹽等。 關於培養，將碳來源或氮來源或磷酸鹽，於最初加入 總量於培養基時，因爲將造成微生物生長發育不良影響， 所以由開始培養後之適當時期部份追加，可提昇r 一亞麻 酸之生產性爲宜。 其他培養條件’例如溫度、培養時間係考量培養基組 成或目的之r -亞麻酸含有率或脂質生產性，設定適宜的 條件即可。溫度通常爲2 0至3 5 °c程度，以2 5至3 0 °c爲 宜’ pH通常爲3至7程度，以3.5至6.5爲宜，通常進行 7 2至2 4 0小時程度，以9 6至1 6 8小時爲宜。 因爲含GLA脂質通常蓄積於微生物菌體中，所以由 常法將培養液固液分離，而得含有含G L A脂質之菌體。 依GLA用途，雖亦可直接使用此菌體，但爲進一步將油 脂萃取精製，以B 1 i g h & D y e r法、F ο 1 i c h法或特開昭5 7 — 1 4 4 9 8 6號公報等所記載之方法，進行菌體破碎、溶劑萃取 ，而可得目的之含GLA油脂。 將如上述所得之含G L A油脂，由已知方法水解處理 或酯交換處理，可得本發明之促進動物細胞增殖用劑油脂 組成物或皮膚外用劑組成物。以如此方法得到本發明之促 進動物細胞增殖用劑油脂組成物或皮膚外用劑組成物時， 促進動物細胞增殖用劑油脂組成物或皮膚外用劑組成物中 之GLA含有比率係採取部份之促進動物細胞增殖用劑油 脂組成物或皮膚外用劑組成物，經甲基酯化後，由氣相層 析所決定（參考特開昭5 7 — 1 449 86號公報）。 200522937 (10) 本發明之促進動物細胞增殖用劑油脂組成物中，只要 非抑制本發明效果者，亦可含有其他成份。作爲其他成份 ’可舉例如GLA以外之上述脂肪酸等。使用來自植物、 來自微生物者爲甘油酯或GLA時，亦可含有來自植物、 來自微生物之其他成份。 本發明之促進動物細胞增殖用劑油脂組成物係依據使 用於培養表皮角化細胞、皮膚纖維母細胞、黑色素細胞、 小腸上皮細胞、人類子宮頸部癌細胞（Hela )及肺纖維母 細胞等之疋者依賴性細胞（a n c h 〇 r a g e d e p e n d e n t c e 11 )、 淋巴球細胞、骨髓腫瘤細胞、白血病細胞及由某細胞與其 他細胞之細胞融合所得之融合瘤細胞（hybridoma)等之動 物細胞，可達成縮短培養時間與增收細胞量。 然而’本發明之促進動物細胞增殖用劑油脂組成物係 只要需要動物細胞增殖之用途，可使用而無限制。可舉例 如動物細胞增殖培養基用添加劑、人工皮膚劑（細胞增殖 用添加劑）、醫藥品、食品及化粧品成份等。以下係說明 關於此等用途。 由添加本發明之促進動物細胞增殖用劑油脂組成物於 動物細胞培養用培養基，可有效率地使動物細胞增殖。可 適用之動物細胞培養用培養基係只要適合於增殖動物細胞 者即可，並無特別的限制，可爲市售基本培養基，亦可爲 將此等適當改變者。作爲具體之基本培養基，可舉例如 MEM ( Minimnn Essential Medium)培養基、Iscove’s 培養 基、RPM 1 1 640 培養基、Ham，s f_12 培養基、Diilbecco，s 200522937 (11) modified Eagle’s medium ( DMEM)培養基、Dulbecco’s modified MEM培養基及加入l〇%FBS之IMDM培養基（ Iscove’s Modified Dulbecco’s Medium)，以 DMEM 培養 基爲宜。此等培養基係可單獨或組合二種以上使用。本發 “ 明之油脂組成物於培養基之添加量係依對象動物細胞種類 . 適當地調整即可。一般添加本發明之促進動物細胞增殖用 劑油脂組成物，使培養液中之濃度成爲0.001至100mg/亳 升程度，以〇·〇1至l〇mg/亳升爲宜，以〇.〇1至img/亳升 _ 尤佳。 加入本發明之促進動物細胞增殖用劑油脂組成物於培 養基時，係以作爲溶液加入爲宜。調製此溶液所使用之溶 媒係以溶解本發明之促進動物細胞增殖用劑油脂組成物， 而且與培養液相溶性爲宜。作爲如此溶媒，可舉例如甲醇 、二甲基亞硕及丙酮等，以丙酮爲宜。此等溶媒係可單獨 或組合二種以上使用。另外，就抑制因所添加溶媒對於動 物細胞影響之觀點上’溶媒係使用對於培養基總量之2質 · 量％以下爲宜。 添加本發明之促進動物細胞增殖用劑油脂組成物於人 工皮膚生產用培養基’或塗佈或噴射於所培養之人工皮膚 ’可促進人工皮膚早期形成或人工皮膚增加。於塗佈或噴 射時，亦可將本發明之促進動物細胞增殖用劑油脂組成物 溶解於適當溶媒之溶液或分散之分散液塗佈或噴射。作爲 溶媒係可使用與上述相同者。人工皮膚生產用培養基時， 溶媒係與上述同樣地，以使用對於培養基總量之2質量％ · -15- 200522937 (12) 以下爲且。另外，上述溶液或分散時，溶媒係於不造成所 培養之人工皮膚不良影響之範圍下，可塗佈或噴霧。 使用本發明之促進動物細胞增殖用劑油脂組成物爲醫 樂品、食品或化粧品成份時，可期待由促進動物細胞增殖 之各種效果。混合本發明之促進動物細胞增殖用劑油脂組 成物於醫樂品等，可使用已知之方法。 於本發明之皮膚外用劑組成物中，只要不抑制本發明 效果者’可含有一般作爲皮膚外用劑所使用之其他成份。 作爲其他成份，可舉例如精製水、醇類、油性物質、抗頭 皮屑劑、殺菌劑、藥效成份、防腐劑、增黏劑、收斂劑、 保濕劑、粉體、香料、乳化安定劑及pH調整劑等。使用 來自植物、來自微生物者爲甘油酯或GLA時，亦可含有 來自植物、來自微生物之其他成份。 作爲醇類’可舉例如乙醇、具有直鏈或支鏈之烷基或 烯基之高級醇類等，作爲油性物質，可舉例如流動鏈烷烴 、凡士林及固體鏈烷烴等之烴類；液狀含水羊毛脂、羊毛 脂脂肪酸等之羊毛脂衍生物；二甲基聚矽氧烷、聚醚變性 聚砂氧烷及胺基變性聚矽氧烷等之聚矽氧烷衍生物、高級 醇高級脂肪酸酯類、高級脂肪酸、具有烷基或烯基之長鏈 醯胺等之油脂類；貂油及橄欖油等之動植物性油脂類等。 作爲藥效成份’可舉例如維生素類等。作爲防腐劑，可舉 例如paraben類等，作爲增黏劑，可舉例如水溶性高分子 化合物等。作爲保濕劑，可舉例如丙二醇、甘油、卡必醇 、3 —甲基—1，3- 丁二醇及糖類等。 -16- 200522937 (13) 作爲本發明之皮膚外用劑組成物之劑型’並無特別的 限制，可直接使用，亦可適用乳霜狀、乳液狀、化粧水狀 、軟膏狀、粉末狀、糊劑、粉末劑、滴下劑、貼付劑及氣 溶膠劑等之通常皮膚外用劑劑型。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.

本發明之皮膚外用劑組成物之使用量係可依用途而適 當調整。例如配合化粧品時，化粧品中之濃度，通常可爲 0.001至100mg/亳升程度，以配合0.01至10mg/亳升爲宜 ，以0.0 1至1 mg/亳升。另外，使用於創傷（包含褥瘡） 治療時，治療藥中之濃度，通常可爲0.001 mg/亳升以上〜 (直接使用），以配合〇.〇1至500mg/亳升爲宜，以0.01 至lOOmg/亳升尤佳。 【實施方式】 實施例 其次，由實施例再詳細地說明本發明，但本發明並不 局限於此等例者。 使用下述實施例及比較例之原料油脂及油脂組成物之 脂肪酸組成係由特開昭5 7 - 1 448 96號公報所記載之方法 而甲基酯化，由氣相層析法測定。另外，構成實施例及比 較例之油脂組成物之甘油酯組成係基於Iatroscan法（J. Jpn. Oil Chem . soc·，V ο 1 3 5 ? N ο . 8 5 625-631(1986))，由組 合薄層層析法（TLC )及氫火焰離子檢測器（FID )之 Iatroscan TH_10型（Iatros社製）而求之。亦即，將1微 升之試樣溶液，滴於矽膠薄層棒（C h r 〇 m a 1 〇 d S ΠΙ，I a t r 〇 s -17- 200522937 (14) 社製）單側後，進行溶媒展開，溶媒展開後，將矽膠薄層 棒安裝於上述分析機TH - 1 0型，以下述條件分析。 試樣溶液濃度：2 0 m g /亳升丙酮 TLC展開溶媒：己烷/二***/醋酸=70/3 0/ 1 (容量比） 展開距離：10.0cm I a t r 〇 s c a η分析條件 氫流量：160亳升/min 空氣流量：2.0亳升/min 掃描速度：30sec/SCAN 偵測器：氫火焰離子檢測器（FID ) 實施例1 (製造油脂組成物）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)

含10 0g之具有如表1所示脂肪酸組成之含GLA油脂 (出光興產（股）製，商品名：GlanoilHGC，GLA : 25.9 質量％ )及l〇〇g之蒸餾水之反應系統，加入20,000單位 之 Rhizopus niveus產生脂肪酶（天野酵素（股）製，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 0,000U/g)，於攪拌下，以 30°C 下反應 4 8小時。 -18- 200522937 (15) 〔表1〕 脂肪酸組成 含有率（質量％) 棕櫚酸 13.5 棕櫚烯酸 2.6 硬脂酸 1.7 油酸 4 1.9 亞油酸 11.7 r -亞麻酸 25.9Newlase 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

添加1 0質量％之氫氧化鈉水溶液於所得之分解反應 物，調整分解反應物之pH爲8至9後，使用200毫升之 二***萃取分解反應物中之油份，水洗，脫水後，除去醚 而得56.4g之油份（油脂組成物）。 使用部份之此油脂組成物，由上述方法進行構成甘油 酯組成分析，另外，甲基酯化部份之油脂組成物後，以氣 相層析法分析脂肪酸組成。其結果如表3所示。另外，於 表3中，例如C1 6 : 0係指碳原子數爲1 6個，無雙鍵之脂 肪酸，C1 8 : 3係指碳原子數爲1 8個，有3個雙鍵之脂肪 酸。 實施例2 (製造油脂組成物） 於100g之與實施例1所使用者相同之含GLA油脂中 ，加入3g水、30g之甘油及30〇,〇〇〇單位之Alcaligenes sp·產生脂肪酶（名糖產業（股）製，lipasePL，90,000U/g -19- 200522937 (16) )，於攪拌下，以3 0 °C反應7 2小時。 添加1 0質量％之氫氧化鈉水溶液於所得之分解反應 物，調整分解反應物之PH爲8至9後，使用200毫升之 二***萃取分解反應物中之油份，水洗，脫水後，除去醚 而得8 7.9 g之油份（油脂組成物）。 使用部份之此油脂組成物’由上述方法進行構成甘油 酯組成分析，另外，甲基酯化部份之油脂組成物後，以氣 相層析法分析脂肪酸組成。其結果如表3所示。 實施例3 (製造油脂組成物） 實施例1中，除了使用具有如表2所示脂肪酸組成之 含GLA油脂（出光興產（股）製，商品名：GlanoilCS， GLA : 7.9質量％ )爲含GLA油脂以外，由與實施例1相 同的酵素處理及萃取而得油脂組成物。 〔表2〕 脂肪酸組成 含有率（質量％ ) 棕櫚酸 3 1.7 棕櫚烯酸 1 .8 硬脂酸 2.6 油酸 40.7 亞油酸 14.2 7 -亞麻酸 7.9 -20- 200522937 (17) 所得之油脂組成物之分析結果如表3所示。 比較例1 (製造油脂組成物） 實施例1中，除了使用未具有GLA酸之紅花油（ SIGMA社製）以取代使用含GLA油脂以外，進行與實施 例1相同的處理，而得油脂組成物。所得油脂組成物之分 析結果如表3所示。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.

〔表3〕 實施例1 實施例2 實施例3 1比較例1 脂肪酸組成 (質量％ ) 棕櫚酸 (C 1 6 : 〇 ) 9.2 15.0 16.6 5.4 棕櫚烯酸 (C 1 6 : 1 ) 2.0 2.3 1 .9 0.4 硬脂酸 (c 1 8 : 〇) 0.0 1 .7 2.2 1.8 油酸 (c 1 8 ： 1 ) 45.8 40.5 47.9 16.7 亞油酸 (C 】8 _· 2 ) 11.7 13.1 17.1 74.9 τ -亞麻酸 (C18:3) 29.4 25.7 12.4 0.0 甘油酯組成 (質量％ ) 三甘油酯： TG 59.2 13.8 50.5 52.3 二甘油酯： DG 37.2 37.9 43.5 33.4 單甘油酯·’ MG 3.6 46.3 6.0 4.3 實施例4 (細胞增殖試驗） 評估實施例1至3及比較例1所得之油脂組成物，對 -21 - 200522937 (18) 於正常人類表皮角化細胞之促進增殖效果。[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.

進行將正常人類表皮角化細胞（三光純藥（股）製， Cryo NHEK—Neo:來自新生兒），使用於 500毫升之 BlettkitKMG基礎培養基（三光純藥（股）製），添加 2 亳升之附屬之牛腦下垂體萃取液、〇 . 5亳升之上皮成長因 子（hEGF)及0.5亳升之胰島素之試驗培養基，於37°C ，5%C02之存在下，培養4至7天，使用成爲70至90% 之未匯合（subconfluent)者於增殖試驗。 進行增殖試驗係使用96孔微培養盤，以細胞密度爲 3.5 X 1 03cell/well，接種正常人類表皮角化細胞（三光純 藥（股）製，Cryo NHEK— Neo :來自新生兒），使用於 5 00毫升之BlettkitKMG基礎培養基（三光純藥（股）製 )，添加2亳升之附屬之牛腦下垂體萃取液、0.025亳升 之上皮成長因子（hEGF)及0.5亳升之胰島素之試驗培養 基，於37°C，5% C02之存在下，培養6天，測定細胞數 細胞數係使用 Premix WST-1 Cell Proliferation Assay System(寶酒造（股）製），以well leader (生化學工業 (股）製，S K 6 0 1型）測定於4 5 0 n m吸收。另外’以不含 本發明之油脂組成物之試驗培養基之細胞數爲基準（1 0 0 % )，算出添加油脂組成物時之增殖率（％ )。結果如表 4所示。 -22- 200522937 (19) 〔表4〕 試料添加量 0.01 mg/ml 0.1 mg/ml 試料 非添加 100% 實施例1 140% 143% 實施例2 13 0% 140% 實施例3 124% 122% 比較例1 104% 106%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%

實施例5 (細胞增殖及提昇抗體生產試驗） 評估實施例1至3及比較例1所得之油脂組成物，對 於CHO細胞（來自中國倉鼠卵細胞之培養細胞）之促進 增殖效果。使用 100亳升之 CHO— S— SFMII培養基（ GIBCOBRL社製），將CHO細胞（ATCC保存株：CRL — 1 1 3 97株），於37°C，5% C02之存在下，靜置培養7天 者’使用於增殖試驗中。 增殖試驗係如下進行。亦即，以0.1 m g /亳升之濃度， 添加各實施例1至3及比較例1所得之油脂組成物於CHO —S — SFMII培養基（GIBCOBRL社製）之10亳升試驗培 養基，懸濁1 X 1 05cell之CHO細胞（ATCC保存株：CRL -1 1 3 97 株），添力口於 INTEGRA CELLINE ( INTEGRA BIOSCIENCES社製，C L — 3 5 0 )中之培養細胞空間。另外 ，將200亳升之未添加上述油脂組成物之CHO - S— SFM Π培養基（GIBCOBRL社製）作爲基礎培養基，添加於培 -23- 200522937 (20) 養細胞空間。 將添加培養基與CH0細胞之上述1NTEGRA CELLINE ，於37°C，5%C〇2之存在下，靜置培養7天後，由培養 液細胞空間回收1 0亳升之細胞懸濁液。將此細胞懸濁液 離心分離（2 0 0 G，5分鐘）’將細胞懸濁於新的試驗培養 基（以0. 1 mg/亳升之濃度，添加各實施例1至3及比較例 1所得之油脂組成物於 CHO — S — SFM Π培養基（ GIBCOBRL社製）之培養基），將此放回培養細胞空間。 之後，繼續於37°C，5% C02之存在下，靜置培養7天。 合計進行1 4天之培養後’回收細胞懸濁液總量。使 用血球計算盤測定此細胞懸濁液中之細胞數後，由酵素結 合免疫吸附法（ELISA法）測定培養上澄液中之IgG抗體 產生量。另外，以僅不含本發明油脂組成物之基礎培養基 (CHO - S — SFM Π培養基（GIBCOBRL社製））進行相同 試驗時之細胞數及IgG抗體產生量爲基準（1〇〇% )，算 出添加油脂組成物時之增殖率（％ )及提昇IgG抗體產生 率（％ )。結果如表5所示。 -24 - 200522937 (21) 〔表5〕 試樣 細胞數 提昇IgG抗體產生 無添加 10 0% 1 0 0 % 實施例1 1 6 3 % 1 9 7 % 實施例2 1 5 4 % 18 5% 實施例3 1 3 3 % 1 6 0 % 比較例1 1 0 8 % 1 1 2 % 實施例6 (治癒效果試驗） 評估使用實施例1至3及比較例1所得之油脂組成物 爲皮膚外用劑組成物時之治癒效果。 將l〇g之甘油、l〇g之流動鏈烷烴及79g之白色凡士 林，加溫至8 0 °C溶解後，冷卻至4 5 °C，添加混合1 g之實 施例1所得之油脂組成物，將此混合物急速冷卻，調製均 勻的軟膏劑。以此爲實施例1軟膏。同樣地，使用實施例 2及3及比較例1所得之油脂組成物，調製實施例2軟膏 及實施例3軟膏及比較例1軟膏。 此等軟膏劑治癒效果係由下述方法評估。亦即，將皮 膚出現老化症狀（雛紋）之5 0至6 0歲女性2 0人，分成4 組’每組各5名’第1組爲實施例丨軟膏，第2組爲實施 例2軟膏’第3組爲實施例3軟膏及第4組爲比較例1軟 霄’使用1個月’依下述評估基準評估使用前後之皮膚皺 紋狀況。評估結果如表6所示。另外，表6之數値係符合 各評估基準之人數。 -25- 200522937 (22) 〈評估基準〉 有效··顯示皺紋明顯消失，表皮張力恢復。 略有效：顯示皺紋變淺，變得不明顯等之改善效果。 無效：使用前後，未見有效果。 〔表6〕 評價 實施例1 實施例2 實施例3 比較例1 軟膏 軟膏 軟膏 軟膏 有效 3 4 1 0 略有效 2 1 3 1 無效 0 0 1 4Example 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

產業上利用性 由利用本發明之促進動物細胞增殖用劑油脂組成物於 促進動物細胞增殖，可達成提昇動物細胞生產性，或提昇 具有有效地產生細胞生理活性物質之生產性。 另外，本發明之皮膚外用劑組成物係具有促進人類及 動物上皮形成之能力，可期待對於由表皮新陳代謝之黑斑 、皺紋對策及治療創傷（包含褥瘡）等之治癒效果者。 -26-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) 十、申請專利範圍 1 · 一種促進動物細胞增殖用劑油脂組成物，其特徵 爲，含三甘油酯、二甘油酯、單甘油酯及游離脂肪酸之油 脂組成物，含一種以上選自r -亞麻酸及r -亞麻酸甘油 酯。 2.如申請專利範圍第1項之促進動物細胞增殖用劑 油脂組成物，其中該三甘油酯、二甘油酯及單甘油酯之含 有比率係對於甘油酯總量，分別爲1 〇至79質量％、20至 70質量％及1至70質量％。 3 .如申請專利範圍第2項之促進動物細胞增殖用劑 油脂組成物，其中該三甘油酯、二甘油酯及單甘油酯之含 有比率係對於甘油酯總量，分別爲1 0至70質量％、25至 60質量％及3至65質量％。 4. 如申請專利範圍第1項至第3頁中任一項之促進 動物細胞增殖用劑油脂組成物，其中r -亞麻酸之含有比 率係對於構成該甘油酯之脂肪酸及游離脂肪酸之合計量爲 5質量％以上。 5. 如申請專利範圍第1項至第4項中任一項之促進 動物細胞增殖用劑油脂組成物，其中由水解處理或酯交換 處理微生物體內油脂或微生物分泌油脂所得者。 6. 如申請專利範圍第5項之促進動物細胞增殖用劑 油脂組成物，其中該微生物爲被孢黴（Mortierella)屬菌 〇 7. 如申請專利範圍第5項之促進動物細胞增殖用劑 -27- 200522937 (2) 油脂組成物，其中該微生物爲毛黴（M u c o r )屬菌。 8 . —種動物細胞培養用培養基，其特徵爲，含有如 申請專利範圍第1項至第7項中任一項之促進動物細胞增 殖用劑油脂組成物。 9. 一種皮膚外用劑組成物，其特徵爲，含三甘油酯 、二甘油酯、單甘油酯及游離脂肪酸之油脂組成物，含有 一種以上選自r 一亞麻酸及r 一亞麻酸甘油酯。 ίο.如申請專利範圍第9項之皮膚外用劑組成物，其 中該三甘油酯、二甘油酯及單甘油酯之含有比率係對於甘 油酯總量，分別爲10至79質量％、20至70質量％及1 至70質量％。 11 .如申請專利範圍第1 0項之皮膚外用劑組成物， 其中該三甘油酯、二甘油酯及單甘油酯之含有比率係對於 甘油酯總量，分別爲1 〇至7 0質量％、2 5至6 0質量％及 3至65質量％。 1 2 .如申請專利範圍第9項至第1 1項中任一項之皮 膚外用劑組成物，其中7 -亞麻酸之含有比率係對於構成 該甘油酯之脂肪酸及游離脂肪酸之合計量爲5質量％以上 〇 1 3 .如申請專利範圍第9項至第1 2項中任一項之皮 膚外用劑組成物，其中由水解處理或酯交換處理微生物體 內油脂或微生物分泌油脂所得者。 1 4 ·如申請專利範圍第〗3項之皮膚外用劑組成物， 其中該微生物爲被孢黴（Mortierella)屬菌。 -28- 200522937 (3) 1 5 .如申請專利範圍第1 3項之皮膚外用劑組成物， 其中該微生物爲毛黴（Miicor )屬菌。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 七 明 說 單 簡 號 為符 圖件 表元 代之 定圖 指表 :案代 圖本本 表' . 代 Z-'N 定一二 指CC 無 無 八、本案若有化學式時，請揭示最能顯示發明特徵的化學 式：無-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