200930358 九、發明說明 【發明所屬之技術領域】 本發明係有關以花生四烯酸、使花生四烯酸爲構成脂 肪酸之化合物、或該等物質之混合物爲有效成份之抗疲勞 劑，以及具有抗疲勞作用之醫藥組成物（包含功能性食品 〇 【先前技術】 疲勞是一種一般主要症狀爲疲勞感、倦怠感，但也伴 隨其他如睡眠障礙及慾望降低等各種症狀之疾病。疲勞感 及倦怠感是提醒身體出現異常的一個重要的警示訊號，在 健康時進行激烈運動、長時間的勞動及處於過度壓力等狀 況時，可自覺出疲勞感及倦怠感。這種生理上的疲勞通常 可藉由讓身體休息而恢復至原本的正常狀態，不會長時間 持續。在1985年總統府所進行的「與健康有關的國民意 〇 識調查」中，約有六成多的人表示感到疲勞，而在表示感 到疲勞的人當中有七成的人回答「藉由一個晚上的睡眠可 恢復疲勞」。然而，進年來該傾向不斷地在改變當中。根 據1 999年所進行之厚生省疲勞調查硏究班所實施之疫學 ^ 調查，報告指出自覺有疲勞的人當中，有六成的人感覺到 疲勞已持續六個月以上。亦即自1985年至19 99年這14 年間，苦於慢性疲勞的人增加，且疲勞的性質也已產生變 化（非專利文件1)。因此目前慢性疲勞症候群（Chronic Fatigue Syndrome ; CFS)所造成的疾病以及過勞死，變成 -5- 200930358 一個相當大的社會問題。然而，與疲勞有關的原因 機轉相當地多，雖有許多學者正在進行硏究當中， 掌握其全貌，且目前尙未確立慢性疲勞症候群及過 起因於疲勞之疾病之明確的治療或預防方法。 最近報告指出一種具有疲勞減輕作用，及可提 勞狀態恢復爲正常狀態作用等，亦即被稱爲「抗疲 」之物質。例如報告有某種胺基酸組成物（專利文件 0 及與左旋肉鹼與組胺酸有關之雙肽（專利文件2) 萃取物（專利文件3)等具體力增強作用》另外， 有目的爲於因運動等而造成體力消耗及疲勞時之營 ，適用之含抗壞血酸之營養補給組成物（專利文件 另外，由本專利申請人揭示了花生四烯酸以及 四烯酸爲構成脂肪酸之化合物，具有預防或改善腦 低以及其所伴隨症狀或疾病之作用（專利文件5 ) 具有改善於日常生活中活動量降低以及/或憂鬱症 〇 用（專利文件6 )。 但並未有任何文獻針對藉由經口投予花生四烯 使花生四烯酸爲構成脂肪酸之化合物之抗疲勞作用 何的暗示或揭示。 專利文件1 :特開平9-1 24473號公報 專利文件2 :特開200 1 -046021號公報 專利文件3 :特開平8-473 8 1號公報 專利文件4 :特開平6-327435號公報 專利文件5:特開20 06-83136號公報 、致病 但仍未 勞死等 早由疲 勞物質 1)， ，山楂 亦揭示 養補給 4 ) ° 使花生 功能降 ，以及 狀之作 酸，或 ，有任 200930358 專利文件6 :特開2 0 0 7 - 8 8 6 1號公報 非專利文件1 :疲勞科學（9.疲勞恢復資訊；200 1， 講談社） 【發明內容】 [發明欲解決之課題] 本發明係以提供因對人及動物等安全，而可持續攝取 〇 ，且對疲勞之預防以及/或治療有效之組成物，特別係醫 藥組成物爲目的。 [解決課題之手段] 本發明之發明者們針對上述課題進行專心檢討後，發 現藉由經口攝取使花生四烯酸爲構成脂肪酸之化合物，可 獲得抗疲勞作用（疲勞之減輕作用以及促進疲勞恢復作用 )，本發明遂至完成。 Ο 亦即，本發明係關於下述之內容。 1. 一種抗疲勞劑，其係以花生四烯酸、使花生四烯 酸爲構成脂肪酸之化合物或該等物質之混合物爲有效成份 〇 2. 如上述1項之抗疲勞劑’其中成人每日投予量以 花生四烯酸換算時爲10〜800mg。 3. 如上述1或2項之抗疲勞劑’其係一週以上之持 續投予。 4. 如上述1〜3項中任一項之抗疲勞劑，其中使花生 200930358 四烯酸爲構成脂肪酸之化合物係花生四烯酸之醇酯、或構 成脂肪酸之一部分或全部爲花生四烯酸之三酸甘油酯、磷 脂質或糖脂質。 5 ·如上述1〜4項中任一項之抗疲勞劑，其係用作治 療或預防疲勞狀態。 6.如上述1〜5項中任一項之抗疲勞劑，其係經口劑 〇 0 7. —種醫藥組成物，其係包含如上述1〜6項中任一 項之抗疲勞劑。 8. —種花生四烯酸、使花生四烯酸爲構成脂肪酸之 化合物或該等物質之混合物之使用，其係爲了製造抗疲勞 劑。 9. 一種方法，其係疲勞狀態之預防或治療之方法， 其係包含對需要其之對象，投予花生四烯酸、使花生四烯 酸爲構成脂肪酸之化合物或該等物質之混合物。 ❾ [發明的效果] 本發明之抗疲勞劑具有優異的疲勞減輕、疲勞恢復促 進作用，且對人類及非人類之動物都很安全，可持續進行 投予。因此，本發明之抗疲勞劑係可做爲包含功能性食品 之醫藥組成物，可廣泛應用。 (花生四烯酸，以及使花生四烯酸爲構成脂肪酸之化合物） 本發明之抗疲勞劑有效成份係花生四烯酸，於本發明 -8- 200930358 之抗疲勞劑中，除游離的花生四烯酸之外，可利用使花生 四烯酸爲構成脂肪酸之全部的化合物（本說明書中將該等 物質總稱爲花生四烯酸類）。使花生四烯酸爲構成脂肪酸 之化合物，並無任何限定，可利用花生四烯酸鹽（例如鈣 鹽、鈉鹽等），及花生四烯酸之低碳醇酯（例如花生四烯 酸甲酯、花生四烯酸乙酯等），及構成脂肪酸之一部分或 全部爲花生四烯酸之三酸甘油酯、磷脂質、糖脂質等。 〇 因本發明的抗疲勞劑之有效成份爲花生四烯酸，以使 用花生四烯酸含量高者爲佳。例如前述之含花生四烯酸之 三酸甘油酯（與構成脂肪酸之一部分或全部爲含花生四烯 酸之三酸甘油酯，或含其之油脂同義），構成三酸甘油酯 之全部脂肪酸中花生四烯酸的比例可舉出以10重量（ W/W) %以上爲佳，20重量％以上更佳，30重量％以上， 40重量％以上之油脂（三酸甘油酯）最佳。該種含花生四 烯酸之三酸甘油酯，除了例如藉由培養具有可生產含花生 〇 四烯酸油脂（三酸甘油酯）之微生物，可進行工業上的製 造，及含25質量％之花生四烯酸之微生物油（含95%以上 之三酸甘油酯）（SUNTGA (註冊商標）25，三多利股份 有限公司），及含40質量％之花生四烯酸之微生物油（含 95%以上之三酸甘油酯）（SUNTGA (註冊商標）40S，三 多利股份有限公司）之外，可根據特開2003-4883 1號公 報，及特開2006-83 1 3 6號公報及 W02003/ 004667號所 記載之方法以及條件，培養具有含花生四烯酸油脂（三酸 甘油酯）生產能力之微生物，而進行製造。具體可使用之 -9- 200930358 微生物、培養條件等請參照該等文獻。 於藉由培養含花生四烯酸油脂之生產菌所生產之含花 生四烯酸油脂（三酸甘油酯）中，提高花生四烯酸比例的 方法’除如前述文獻所記載之控制培養基碳素來源濃度以 外，亦可對含花生四烯酸油脂進行選擇性的水解，而獲得 含高量花生四烯酸之油脂。該用於選擇性水解的解脂酵素 並非具有三酸甘油酯之位置特異性，而因水解活性會與雙 〇 鍵數目成比例而降低，而使高度不飽和脂肪酸以外的脂肪 酸之酯鍵結被水解。因此，於所生成之PUFA部分甘油酯 間引起酯交換反應等，而成爲高度不飽和脂肪酸被提高之 三酸甘油醋（「Enrichment of Archidonic Acid : Selective Hydrolysis of a Single-Cell Oil from Mortierella with Candida cylindracea Lipase」：J· Am. Oil. Chem. Soc.， 72, 1323-1327, 1995)。 如此，可將上述對含花生四烯酸油脂進行選擇性的水 〇 解後所得之含有高花生四烯酸油脂（三酸甘油酯），做爲 本發明之有效成份。相對於本發明之含花生四烯酸油脂（ 三酸甘油酯）之全部脂肪酸，花生四烯酸之比例由於目的 在於排除其他脂肪酸之影響而以較高者爲佳，但並未限定 於高的比例。實際上應用於食品上時，有時花生四烯酸的 絕對含量會成爲問題，實際上可使用含10重量％以上花生 四烯酸油脂（三酸甘油酯）。 (抗疲勞作用以及抗疲勞劑） -10- 200930358 藉由將上述之花生四烯酸類對人類以及非 行經口投予，可獲得顯著的抗疲勞作用。此處 係指做爲抗疲勞對象之動物，而本發明之抗疲 用於感覺疲勞之人類、畜產動物、寵物以及實 別適用於人類。畜產動物係指牛、馬、豬、山 家畜及賽馬、獵犬等，寵物則指狗、貓、土撥 ，實驗動物爲小鼠、大鼠、天竺鼠、米格魯犬 〇 普通獼猴、長尾獼猴等提供醫學、生物學、農 領域硏究之動物。本發明之抗疲勞劑，可使用 之人類、畜產動物、寵物以及實驗動物，特別 〇 疲勞係指連續加諸於身體上或精神上負荷 的一時性身體以及精神表現降低之現象，表現 體以及精神的工作能力的質或量降低。於本發 」中亦包含慢性疲勞症候群及過勞死。200930358 IX. INSTRUCTIONS OF THE INVENTION [Technical Field] The present invention relates to an anti-fatigue agent which uses arachidonic acid, a compound which makes arachidonic acid as a constituent fatty acid, or a mixture of such substances as an active ingredient, and has an anti-fatigue agent Fatigue action pharmaceutical composition (including functional foods [previous technique] Fatigue is a general symptom of fatigue, burnout, but also other diseases such as sleep disorders and decreased desire. Fatigue and burnout It is an important warning signal to remind the body of abnormalities. When exercising intensely during exercise, prolonged labor, and being overstressed, it can feel fatigue and burnout. This physical fatigue can usually be relied on. Let the body rest and return to the original normal state, it will not last for a long time. In the "Health-related public opinion survey" conducted by the Presidential Palace in 1985, about 60% said that they feel tired, but Seventy percent of those who feel tired are replied, "Restoring fatigue through one night's sleep." However, this tendency has been constantly changing over the years. According to the epidemiological survey conducted by the Ministry of Health and Welfare Survey in the Year of 1999, the report pointed out that 60% of those who feel fatigued feel it. Fatigue has lasted for more than six months. That is, during the 14 years from 1985 to 1999, the number of people suffering from chronic fatigue has increased, and the nature of fatigue has also changed (Non-Patent Document 1). Therefore, the current chronic fatigue syndrome (Chronic) The disease caused by Fatigue Syndrome (CFS) and overwork death have become a considerable social problem. - However, the causes related to fatigue are quite numerous, although many scholars are conducting research, mastering It has a complete picture, and there is no clear treatment or prevention method for chronic fatigue syndrome and disease caused by fatigue. Recently, it has been reported that it has a fatigue-reducing effect and the recovery of the state of recovery to a normal state, that is, a substance called "anti-wear". For example, it is reported that there is an amino acid composition (Patent Document 0 and with L-carnitine and histidine Bi-peptide (Patent Document 2) Extracts (Patent Document 3) and other specific force enhancement effects. In addition, there is a purpose to provide a nutrient replenishing composition containing ascorbic acid for camps that cause physical exertion and fatigue due to exercise or the like ( Patent Document In addition, the applicant of the present patent discloses that arachidonic acid and tetraenoic acid are compounds constituting a fatty acid, and have the effect of preventing or improving brain low and its accompanying symptoms or diseases (Patent Document 5) has been improved in daily life. Reduced activity and/or depression (Patent Document 6). However, there is no suggestion on the anti-fatigue effect of arachidonic acid as a compound constituting a fatty acid by oral administration of arachidonic acid. Patent Document 1: Japanese Laid-Open Patent Publication No. Hei No. Hei. No. Hei. No. Hei. No. Hei. No. Hei. No. Hei. Patent Document 5: JP-A 20 06-83136, causing illness, but still not dying, such as early fatigue material 1), Hawthorn also revealed that feeding to 4) ° to reduce the function of peanuts, and Acidic, or, 200930358 Patent Document 6: Special Open 2 0 0 7 - 8 8 6 1 Non-Patent Document 1: Fatigue Science (9. Fatigue Recovery Information; 200 1, Kodansha) [Summary] [Problem to be Solved by the Invention] The present invention is directed to a composition which is effective for prevention and/or treatment of fatigue due to safety of humans and animals, and is particularly effective as a pharmaceutical composition. [Means for Solving the Problem] The inventors of the present invention have conducted an intensive review of the above-mentioned problems and found that arachidonic acid is a compound constituting a fatty acid by oral ingestion, thereby obtaining an anti-fatigue effect (a reduction effect of fatigue and promotion of fatigue). Recovery effect), the present invention is completed. That is, the present invention relates to the following. An anti-fatigue agent which comprises arachidonic acid, a compound which makes arachidonic acid as a constituent fatty acid or a mixture of such substances as an active ingredient 〇 2. The anti-fatigue agent of the above item 1 wherein the adult daily The dose is 10 to 800 mg in terms of arachidonic acid. 3. The anti-fatigue agent of item 1 or 2 above is continuously administered for more than one week. 4. The anti-fatigue agent according to any one of items 1 to 3 above, wherein the peanut 200930358 tetraenoic acid is a compound constituting a fatty acid, an alcohol ester of arachidonic acid, or a part or all of a constituent fatty acid is arachidonic acid. Triglyceride, phospholipid or glycolipid. The anti-fatigue agent according to any one of items 1 to 4 above, which is used for treating or preventing fatigue. 6. The anti-fatigue agent according to any one of items 1 to 5 above, which is an oral medicinal composition comprising the anti-fatigue agent according to any one of items 1 to 6 above. 8. Use of arachidonic acid, arachidonic acid as a compound constituting a fatty acid or a mixture of such materials for the manufacture of an anti-fatigue agent. A method for preventing or treating a fatigue state, which comprises administering to a subject in need thereof, arachidonic acid, arachidonic acid as a compound constituting a fatty acid or a mixture of such substances. ❾ [Effects of the Invention] The anti-fatigue agent of the present invention has excellent fatigue reduction, fatigue recovery promotion, and is safe and sustainable for human and non-human animals. Therefore, the anti-fatigue agent of the present invention can be used as a pharmaceutical composition containing a functional food and can be widely applied. (Arachidonic acid, and a compound which makes arachidonic acid a constituent fatty acid) The anti-fatigue agent active ingredient of the present invention is arachidonic acid, and the anti-fatigue agent of the present invention -8-200930358, except for the free peanut four In addition to the enoic acid, arachidonic acid can be used as a compound constituting all of the fatty acids (the substances in the present specification are collectively referred to as arachidonic acid). The arachidonic acid is a compound constituting the fatty acid, and is not limited thereto, and an arachidonic acid salt (for example, a calcium salt, a sodium salt, etc.) and a lower alcoholic acid of arachidonic acid (for example, arachidonic acid A) can be used. Ester, arachidonic acid ethyl ester, etc., and some or all of the constituent fatty acids are arachidonic acid triglycerides, phospholipids, glycolipids and the like. 〇 Since the active ingredient of the anti-fatigue agent of the present invention is arachidonic acid, it is preferred to use a high arachidonic acid content. For example, the aforementioned arachidonic acid-containing triglyceride (some or all of the constituent fatty acids is arachidonic acid-containing triglyceride or synonymous with the same), constitutes all fatty acids of the triglyceride The ratio of arachidonic acid is preferably 10 parts by weight or more, more preferably 20% by weight or more, more preferably 30% by weight or more, and 40% by weight or more of the oil or fat (triglyceride). The arachidonic acid-containing triglyceride can be industrially produced, for example, by culturing a microorganism capable of producing a peanut-containing arachidonic acid oil (triglyceride), and contains 25% by mass. Arachidonic acid microbial oil (containing more than 95% triglyceride) (SUNTGA (registered trademark) 25, Sandoli Co., Ltd.), and microbial oil containing 40% by mass of arachidonic acid (including 95 In addition to the above-mentioned triglyceride (SUNTGA (registered trademark) 40S, Sandoli Co., Ltd.), it can be issued according to JP-A-2003-4883, and JP-A-2006-83 1 3 6 and W02003. The method and conditions described in No. 004667 are carried out by culturing a microorganism having an arachidonic acid-containing fat (triglyceride)-producing ability. Specific use -9- 200930358 Please refer to these documents for microorganisms, culture conditions, etc. A method for increasing the ratio of arachidonic acid in an arachidonic acid-containing fat (triglyceride) produced by cultivating a production strain containing arachidonic acid oil, except for controlling medium carbon as described in the aforementioned document In addition to the source concentration, the arachidonic acid-containing fat or oil can be selectively hydrolyzed to obtain a high-amount arachidonic acid-containing fat. The lipolytic enzyme for selective hydrolysis does not have the positional specificity of triglyceride, and the hydrolysis activity is reduced in proportion to the number of biguanide bonds, and the ester bond of fatty acids other than highly unsaturated fatty acids is hydrolysis. Therefore, a transesterification reaction or the like is caused between the generated glycerides of the PUFA portion, and the glycerol vinegar is highly enhanced. (Enrichment of Archidonic Acid: Selective Hydrolysis of a Single-Cell Oil from Mortierella with Candida cylindracea Lipase": J. Am. Oil. Chem. Soc., 72, 1323-1327, 1995). Thus, the above-mentioned arachidonic acid-containing fat or oil can be selectively hydrolyzed to obtain a high arachidonic acid oil (triglyceride) as an active ingredient of the present invention. The ratio of arachidonic acid to the total fatty acid of the arachidonic acid-containing fat (triglyceride) of the present invention is preferably higher because it is intended to exclude the influence of other fatty acids, but is not limited to high. proportion. In fact, when applied to foods, the absolute content of arachidonic acid may become a problem, and in fact, an arachidonic acid oil (triglyceride) containing 10% by weight or more may be used. (Anti-fatigue effect and anti-fatigue agent) -10- 200930358 By administering the above-mentioned arachidonic acid to humans and non-administered oral administration, significant anti-fatigue effects can be obtained. Here, it refers to an animal which is an object of anti-fatigue, and the anti-fatigue of the present invention is used for humans, animal animals, pets, and practically suitable for human beings. Animals refer to cattle, horses, pigs, mountain livestock and horse racing, hounds, etc. Pets refer to dogs, cats, and earthworms. Experimental animals are mice, rats, guinea pigs, Miguel dogs, common macaques, long-tailed macaques, etc. Provide animals in the fields of medicine, biology and agriculture. The anti-fatigue agent of the present invention can be used for humans, livestock animals, pets, and experimental animals, and particularly fatigue refers to a phenomenon in which the body is physically and mentally loaded, and the mental performance is reduced, the expression and the spirit are reduced. The quality or quantity of work ability is reduced. Chronic fatigue syndrome and overwork death are also included in this issue.
〇 本發明中「抗疲勞作用」，亦即「抗疲勞I 可稱其爲具有可使前述之疲勞減弱及使疲勞恢 具體而言，係具有提升運動及作用部位（包含 的持續時間，以及於相同的運動量及工作量之 疲勞物質的增加（持久力提升.體力增強）， 工作部位是否疲勞，改善腦及神經等所呈現的 態，以及促進運動及工作部位自疲勞狀態恢復 之效果。 本發明抗疲勞劑目標之慢性疲勞症候群， 人類動物進 非人類動物 勞劑，可使 驗動物，特 羊、綿羊等 鼠、倉鼠等 、迷你豬、 學、藥學等 於感覺疲勞 適用於人類 時，感受到 降低意指身 明之「疲勞 ftf」之效果， 復之作用， 腦部）工作 下，可抑制 無論運動及 疲勞感知狀 至一般狀態 係指成爲曰 -11 - 200930358 常生活障礙之長期性的全身疲勞感、倦怠感、輕微發燒、 淋巴結腫脹、肌肉疼痛、關節疼痛、精神方面的症狀等基 本症狀。本發明之抗疲勞劑可處理該慢性疲勞症候群，亦 即可緩和慢性疲勞症候群之各種症狀，而可使其轉變至正 常狀態。本發明之抗疲勞劑目標之過勞死係指處於重度過 勞狀態，無論是否無法保持身體的活力，均無法確實感覺 到疲勞，其結果，發生腦血管疾病及心臟疾病症狀，導致 〇 永久無法工作及死亡狀態。本發明之抗疲勞劑可處理慢性 疲勞症候群，更可預防過勞死。 本發明之「抗疲勞劑」之效果，可藉由例如於浸水中 斷睡眠試驗中測定游泳時間而進行評價。該試驗如同進行 浸水飼育，使用於無法獲得充分睡眠及休息姿勢，肉體及 精神均處於無法休息之環境下所飼育之小鼠，迫使其於負 載重錘之狀態下游泳，藉由測定力竭游泳時間（例如以最 初鼻子沒入水中1 0秒以上開始至完全沉入水中的時間， © 或於最後（至無法再浮上來爲止）鼻頭沒入水中時至完全 沉入水中之時間），可確認其疲勞度。而因該動物模型爲 肉體及精神的疲勞模型，藉由投予被試驗物質可延長游泳 時間時，表示身體上及/或精神上的疲勞被減輕，還有疲 勞狀態下身體的活力被維持（體力被增強），提升持久力 等，確認其對疲勞的抵抗性。 抗疲勞劑之效果除上述之浸水中斷睡眠試驗之外，可 藉由於強制運動時測定自發運動量，以及測定血中疲勞物 質量等加以確認。 -12- 200930358 本發明之發明者們檢討結果，確認使小鼠經口攝取使 花生四烯酸爲構成脂肪酸之化合物（特別係含花生四烯酸 之三酸甘油酯）時，於前述之浸水中斷睡眠試驗中，對疲 勞具有抵抗性。該結果不僅意味花生四烯酸可做爲抗疲勞 劑，亦適用於慢性疲勞症候群之預防及治療，及預防過勞 死。 如上所述，可藉由服用本發明之抗疲勞劑而不易疲勞 0 ，或是具有疲勞恢復效果。亦即於進行體育運動等肌肉運 動而感覺肉體疲勞時，及計算工作等連續工作而感覺精神 疲勞時可進行服用而可以期待疲勞恢復效果，而預先服用 再進行勞動、體育運動等時可預防疲勞產生。另外可藉由 於進行體育運動之前及中途進行攝取，而可期待增加持久 力。進而，藉由日常性地攝取，亦可預防精神性的疲勞及 其所伴隨之疾病。 本發明之抗疲勞劑可使用爲醫藥品及食品（功能性食 φ 品、健康輔助食品、營養功能食品、特殊用途食品、特定 保健用食品等可經口投予者）。本發明之抗疲勞劑亦可使 用爲於包裝、容器或說明書上表示有效成份之種類、製劑 之用途、提升持久力以及抗疲勞效果等效能’以及/或攝 取方法。 將本發明之疲勞劑使用爲醫藥品時，可單獨將花生四 烯酸以及/或使花生四烯酸爲構成脂肪酸之化合物’或是 與藥理學上所容許之載體'稀釋劑或賦型劑一同’以液劑 、錠劑、顆粒劑、散劑、膠囊劑、糖漿劑或九劑等劑型進 -13- 200930358 行經口投予。投予量及投予型態可視對象、病況及其進行 狀態、其他條件而適當地加以選擇，例如可選擇使花生四 烯酸爲構成脂肪酸之化合物之含花生四烯酸的三酸甘油酯 ，以人類（成人）爲對象，以獲得抗疲勞作用爲目的而進 行經口投予時，所換算之花生四烯酸量以每日10〜800mg 爲佳，50〜700mg更佳，最佳爲攝取100〜500mg左右，1 日1〜3次的頻率連續進行投予。特別係1星期以上，以2 0 星期以上之連續投予爲佳而可獲得抗疲勞作用。且本發明 之發明者們使用小鼠的疲勞動物模型進行檢討後，確認了 希望以預先服用含花生四烯酸油脂而減輕精神上及肉體上 疲勞時，以 10〜800mg/kg’ 50〜700mg/kg更佳，100〜 500mg/kg最佳連續服用2星期，可獲得抗疲勞作用。 將本發明之抗疲勞劑使用爲飲食品時，其搭配量因使 花生四烯酸爲構成脂肪酸之化合物之種類等而異，例如使 用含花生四烯酸之三酸甘油酯時，花生四烯酸以0.001重 〇 量％以上爲佳，0.01重量％以上更佳，〇·1重量％以上爲最 佳之量進行搭配。 本發明之抗疲勞劑若於不會損及花生四烯酸效果的範 圍內，亦即不會因爲與花生四烯酸搭配而產生不希望的交 互作用之範圍內，可因應需要，與其他生理活性成份、礦 物質類、維生素類、荷爾蒙、營養成份、呈味料、香料等 其他添加物進行混合。該等添加物可使用均用於一般醫藥 、食品者。 -14- 200930358 【實施方式】 [實施例] 以下藉由實施例更具體說明本發明’但本發明並未因 此而有所限定。 '實施例1.因攝取花生四烯酸之抗疲勞作用（1) 做爲被試驗物質之花生四烯酸係使用SUNTGA (註冊 Q 商標）40S (含48%花生四烯酸）《SUNTGA (註冊商標） 40S係以特開2003-4883 1號公報（實施例Ο所記載之方 法而製造。 藉由浸水中斷睡眠試驗評價對疲勞之效果。評價方法 係使用將 Tanaka氏等人之方法（Neurosience, Let.3 52, 159-162, 2 00 3)改變一部分之方法而實施。受試動物係使 用Balb/c系雄性小鼠（5週大）（自日本SLC公司購入） ，於試驗環境將其進行馴化一週後，將呈現順利發育之動 Ο 物用於試驗。以使平均體重均等而分爲5群（每群6〜10 隻），以經添加含花生四烯酸油脂之實驗動物用標準飼料 (AIN-9 3G飼料）進行2星期之飼育。之後，將4群（群 2〜群5)爲浸水中斷睡眠壓力群，取代墊料（紙屑）的是 水溫爲23 °C之自來水，且使水深爲7mm並將其放入飼育 籠中’藉此進行小鼠浸水中斷睡眠試驗。浸水中斷睡眠試 驗中以實驗動物用標準飼料（AIN-93G飼料）進行鎖食， 對群3〜群5等3群以前述之將含花生四烯酸油脂溶解於 橄欖油後之飼料，做爲受試檢體，1日丨次，進行2天的 -15- 200930358 強制經口投予（浸水飼育花生四烯酸群）。對照群（群2 )則以橄欖油強制經口投予。剩下的1群（群1 )以普通 的墊料（紙屑）於飼育籠中進行飼育，1天1次，以橄欖 油強制經口投予（普通飼育對照群）。 ❹ 浸水飼育或普通飼育2天後，於小鼠的尾部綁上相當 其體重8 %的重錘，並強迫其於水槽中（直徑18cm，水深 3 0cm )游泳，並測定力竭游泳時間（最後（無法再浮上來 爲止）鼻頭沒入水中，其至沉沒的時間）。浸水飼育群之 小鼠較普通飼育群之小鼠游泳時間縮短，但藉由投予被試 驗檢體（花生四烯酸），藉由游泳時間縮短可被抑制之程 度，來判定對疲勞之效果。 Ο 表1之花生四烯酸量， 生四烯酸油脂添加飼料之量 攝取量，以及自對小鼠所投 予量，而計算之花生四烯酸 表1 群1 (com) —般飼育對照群 群2浸水飼育對照群 群3浸水飼育花生四烯酸群 群4浸水飼育花生四烯酸群 群5浸水飼育花生四烯酸群 係記載自經小鼠所攝取之含花 ，所計算之花生四烯酸的一曰 予之含花生四烯酸油脂量之投 一曰攝取量。 橄欖油 橄欖油 花生四嫌酸224mg/kg/day(橄欖油） 花生四稀酸448mg/kg/day(橄欖油） 花生四稀酸896mg/kg/day(橄横油） 結果如圖1所不。圖1中**係表不根據Student T Test危險率爲o.oi %之有意義差異。如圖i可明確得知， 浸水飼育群對照群之游泳時間，較一般飼育群相比爲有意 義的縮短。反之，可發現於投予被試驗檢體之花生四烯酸 -16- 200930358 224mg/kg ' 448mg/kg、8 96mg/kg之群，特別係投予花生 四烯酸224mg/kg、448mg/kg之群中，游泳時間的短縮被 抑制。該結果暗示藉由經口攝取花生四烯酸可獲得抗疲勞 作用。 實施例2.因攝取花生四烯酸之抗疲勞作用（2) 除如表2所示使各群爲9〜10隻小鼠，並分爲3群之 0 外，與實施例1相同地測定藉由花生四烯酸之抗疲勞作用 。但實施例2中之力竭游泳時間係測定最初鼻頭沒入水中 1 〇秒以上開始，至其完全沉入水中之時間。且表2中花生 四烯酸量係與實施例1以相同方式計算。 表2 群1 (cont) —般飼育對照群 橄横油 群2 浸水飼育對照群橄_ 群3 浸水飼育 花生四烯酸群花生四烯酸112mg/kg/day(橄橫油）「 "Anti-fatigue effect" in the present invention, that is, "anti-fatigue I" can be said to have the above-mentioned fatigue reduction and fatigue recovery, and has the lifting motion and the action site (including the duration, and The same amount of exercise and the increase of the fatigue amount of the workload (resistance increase, physical strength), whether the work site is fatigued, improve the state of the brain and nerves, and promote the recovery of the exercise and the working site from the fatigue state. Anti-fatigue agent target chronic fatigue syndrome, human animals into non-human animal labor, can make animals, special sheep, sheep and other rats, hamsters, etc., mini pigs, learning, pharmacy equal to feeling fatigue, apply to humans, feel lower It means the effect of the "fatigue ftf" of the body, and the function of the brain, under the work of the brain, can suppress the long-term general fatigue of the dysfunction of life from 曰-11 to 200930358. , burnout, mild fever, swollen lymph nodes, muscle pain, joint pain, mental symptoms, etc. Symptoms. The anti-fatigue agent of the present invention can treat the chronic fatigue syndrome, and can alleviate various symptoms of the chronic fatigue syndrome, and can be converted to a normal state. The overwork of the anti-fatigue agent of the present invention means that the patient is in a state of severe overwork, and whether or not the vitality of the body cannot be maintained, the fatigue cannot be surely felt. As a result, cerebrovascular diseases and symptoms of heart disease occur, resulting in permanent failure of the sputum. Work and death status. The anti-fatigue agent of the present invention can treat chronic fatigue syndrome and prevent overwork death. The effect of the "anti-fatigue agent" of the present invention can be evaluated by, for example, measuring the swimming time in a water-immersed sleep test. The test is like a soaking in water, which is used for mice that are unable to obtain adequate sleep and rest posture, and that are both physically and mentally unable to rest, forcing them to swim under the load of a heavy hammer, and to measure exhaustive swimming. Time (for example, the time from the initial nose to the water for more than 10 seconds to the time of complete submersion, © or at the end (to the time when it is no longer possible to float) when the nose is submerged into the water until it is completely submerged) Its fatigue. Since the animal model is a physical and mental fatigue model, when the test substance is administered to prolong the swimming time, it means that the physical and/or mental fatigue is alleviated, and the vitality of the body is maintained under fatigue ( Physical strength is enhanced), and the endurance is improved to confirm its resistance to fatigue. The effect of the anti-fatigue agent can be confirmed by measuring the amount of spontaneous exercise during forced exercise and measuring the amount of fatigue in the blood, in addition to the above-described water immersion interruption sleep test. -12- 200930358 The inventors of the present invention reviewed the results and confirmed that when the mice were orally ingested to make arachidonic acid a compound constituting a fatty acid (particularly a triglyceride containing arachidonic acid), the water was soaked in the foregoing. In the interrupted sleep test, it is resistant to fatigue. This result not only means that arachidonic acid can be used as an anti-fatigue agent, but also for the prevention and treatment of chronic fatigue syndrome and prevention of overwork. As described above, it is possible to prevent fatigue by taking the anti-fatigue agent of the present invention, or to have a fatigue recovery effect. When you feel physical fatigue such as sports and physical fatigue, you can take it while you are feeling tired due to continuous work such as calculation work. You can expect fatigue recovery effect, and prevent fatigue when you take it beforehand for labor or sports. produce. In addition, it is expected to increase the endurance by taking in before and during sports. Further, by daily ingestion, it is possible to prevent mental fatigue and the accompanying diseases. The anti-fatigue agent of the present invention can be used as a pharmaceutical or food (functional food product, health supplement food, nutritious function food, special purpose food, and special health food, etc.). The anti-fatigue agent of the present invention can also be used to indicate the type of the active ingredient, the use of the preparation, the durability and the anti-fatigue effect equivalent energy' and/or the method of taking in a package, container or instructions. When the fatigue agent of the present invention is used as a pharmaceutical, arachidonic acid and/or arachidonic acid may be used alone as a compound constituting a fatty acid or a pharmacologically acceptable carrier 'diluent or excipient Together with the liquid agent, lozenge, granules, powder, capsules, syrup or nine doses into the injection -13-200930358 by oral administration. The dosage and the dosage form can be appropriately selected depending on the subject, the condition, the state of progress, and other conditions. For example, arachidonic acid-containing triglyceride which is a compound constituting the fatty acid can be selected, When human (adult) is used for oral administration for the purpose of anti-fatigue, the amount of arachidonic acid converted is preferably 10 to 800 mg per day, more preferably 50 to 700 mg, and most preferably ingested. It is administered continuously at a frequency of about 100 to 500 mg at a frequency of 1 to 3 times a day. In particular, for more than one week, anti-fatigue effects can be obtained by continuous administration for more than 20 weeks. Further, the inventors of the present invention examined the fatigue animal model of the mouse and confirmed that it is desirable to reduce the mental and physical fatigue by taking the arachidonic acid-containing fat in advance, and to use 10 to 800 mg/kg '50 to 700 mg. /kg is better, 100~500mg/kg is best taken continuously for 2 weeks, and anti-fatigue effect can be obtained. When the anti-fatigue agent of the present invention is used as a food or drink, the amount of the anti-fatigue agent varies depending on the type of the compound which constitutes the fatty acid, for example, when arachidonic acid-containing triglyceride is used, arachidene The acid is preferably 0.001% by weight or more, more preferably 0.01% by weight or more, and more preferably 〇1% by weight or more. The anti-fatigue agent of the present invention can be used in a range that does not impair the effect of arachidonic acid, that is, it does not cause an undesirable interaction with arachidonic acid, and can be adapted to other physiological conditions as needed. Mixing active ingredients, minerals, vitamins, hormones, nutrients, flavorings, spices and other additives. These additives can be used in general medicines and foods. [Embodiment] [Embodiment] Hereinafter, the present invention will be described more specifically by way of examples, but the invention is not limited thereto. 'Example 1. Anti-fatigue effect by arachidonic acid intake (1) As a test substance, arachidonic acid is used SUNTAGA (registered Q trademark) 40S (including 48% arachidonic acid) "SUNTGA (registered 40S is manufactured by the method described in JP-A-2003-4883 (Example Ο. The effect of fatigue is evaluated by the water immersion interruption sleep test. The evaluation method uses the method of Tanaka et al. (Neurosience, Let.3 52, 159-162, 2 00 3) Implemented by changing the method. The test animals were treated with Balb/c male mice (5 weeks old) (purchased from SLC Japan) and tested in the test environment. After one week of acclimation, the animals that developed smoothly developed were used for the experiment. The average body weight was equally divided into 5 groups (6 to 10 per group), and the standard for experimental animals containing arachidonic acid and fat was added. Feed (AIN-9 3G feed) was bred for 2 weeks. After that, 4 groups (Group 2 to Group 5) were immersed in water to interrupt the sleep pressure group, and instead of litter (paper scrap), tap water with a water temperature of 23 °C was used. And make the water depth 7mm and put it in the breeding cage' The rats were immersed in water and interrupted the sleep test. In the immersion interruption sleep test, the experimental animals were used to lock the food with the standard feed (AIN-93G feed), and the group 3 to the group 5 and the like were dissolved in the olive oil containing the arachidonic acid oil as described above. After the feed, as the test sample, 1 day, 2 days of -15-200930358 forced oral administration (immersion in the arachidonic acid group). Control group (group 2) was forced with olive oil Oral administration. The remaining group 1 (group 1) was bred in a feeding cage with ordinary litter (paper scraps), once a day, and forcedly orally administered with olive oil (common breeding control group). Two days after soaking or normal breeding, a weight of 8% of the weight of the mouse was tied to the tail of the mouse, and forced to swim in the sink (18 cm in diameter, water depth 30 cm), and the exhaustive swimming time was determined (final ( The nose can no longer float up, and the nose is not in the water until it sinks.) The mice in the water-immersed breeding group have shorter swimming time than the mice in the normal breeding group, but by administering the test sample (arachidonic acid) By the shortening of the swimming time, the degree of suppression can be suppressed. To determine the effect on fatigue. Ο The amount of arachidonic acid in Table 1, the amount of feed of retinoic acid and fat added to the feed, and the amount administered from the mice, and the calculated arachidonic acid table 1 group 1 (com) general breeding control group 2 water immersion control group 3 immersed in water arachidonic acid group 4 immersed in water arachidonic acid group 5 immersed in water arachidonic acid group recorded in the mice Containing flowers, the calculated amount of arachidonic acid is given to the amount of arachidonic acid containing oil. Olive oil, olive oil, peanut, four acids, 224mg/kg/day (olive oil), peanut, tetrabasic acid, 448mg /kg/day (olive oil) Peanut tetra-acid 896mg/kg/day (European oil) The results are shown in Figure 1. The ** line in Figure 1 does not differ according to the Student T Test hazard rate of o. oi %. As can be clearly seen in Figure i, the swimming time of the water-immersed breeding group control group is meaningfully shortened compared with the general breeding group. On the contrary, it can be found in the group of arachidonic acid-16-200930358 224mg/kg '448mg/kg, 8 96mg/kg administered to the test subject, especially the arachidonic acid 224mg/kg, 448mg/kg. In the group, the shortening of the swimming time is suppressed. This result suggests that anti-fatigue effects can be obtained by orally ingesting arachidonic acid. Example 2. Anti-fatigue action by arachidonic acid ingestion (2) Measurement was carried out in the same manner as in Example 1 except that each group was 9 to 10 mice as shown in Table 2 and divided into 3 groups of 0. By the anti-fatigue effect of arachidonic acid. However, the exhaustive swimming time in Example 2 was measured from the time when the nose was submerged in water for more than 1 second, until it was completely submerged. And the amount of arachidonic acid in Table 2 was calculated in the same manner as in Example 1. Table 2 Group 1 (cont) - General breeding control group Olive oil group 2 Immersion water breeding control group olives _ group 3 water soaking arachidonic acid group arachidonic acid 112mg/kg/day (diet oil)
❹ 結果如圖2所示。圖2中**係表示根據Student T❹ The result is shown in Figure 2. Figure 2 shows the ** based on Student T
Test危險率爲0.01 %之有意義之差異。如圖2可明確得知 ，浸水飼育群對照群之游泳時間，與一般飼育群相比爲有 意義地縮短。攝取被試驗檢體之花生四烯酸之群中，可發 現游泳時間的縮短抑制效果，確認即使攝取l〇〇mg/kg亦 可獲得抗疲勞作用。 實施例3 .製劑例 (製劑例1 :調製膠囊劑） -17- 200930358 於100質量份之明膠以及35質量份之食品添加物用 甘油中加入水，於50〜60 °C下進行溶解，調製黏度爲 2 00 0cp之明膠被膜。再將含花生四烯酸油脂（SUNTGA ( 註冊商標）40S )，與0.05質量％之維生素E油混合，調 製爲內容物。使用該明膠被膜與內容物，遵照常用方法進 行膠囊成形及乾燥，製成每粒含有內容物爲180 mg之軟 質膠囊。 ❹ (製劑例2 :調製脂肪輸液劑） 加入400g之含花生四烯酸油脂（SUNTGA (註冊商標 )40S) ，48g之純化卵磷脂，20g之油酸，l〇〇g之甘油 以及40ml之0.1N之苛性鈉，以均質混合機使其分散後， 加入注射用蒸餾水至4公升。再使用高壓噴霧式乳化機進 行乳化，調製成脂質乳液。再將該脂質乳液每200ml分注 入塑膠製包裝袋後，再進行121 °C、20分鐘之高壓蒸汽滅 〇 菌處理而製成脂肪輸液劑。 (製劑例3 :調製果汁） 將2g之β-環糊精添加於20ml之20%乙醇水溶液，以 攪拌子攪拌其之同時，加入1 〇〇mg之含花生四烯酸油脂（ SUNTGA (註冊商標）40S中搭配有0.05質量％之維生素 E)，於50°C下進行2小時赙育。冷卻至室溫（約1小時 )後，再持續進行攪拌同時於4°c下進行10小時游育。將 所生成之沉澱以離心分離進行回收，以正己烷洗淨後再進 -18- 200930358 行冷凍乾燥，而得l_8g之含有含花生四烯酸之三酸甘油 酯之環糊精晶籠化合物。取lg該粉末使其與500ml果汁 均勻混合，而調製成含有含花生四烯酸之三酸甘油酯之果 汁。 【圖式簡單說明】 [圖1]係表示於實施例1中’因攝取花生四烯酸之抗 疲勞作用圖。 [圖2]係表示於實施例2中’因攝取花生四烯酸之抗 疲勞作用圖。The test risk rate is a meaningful difference of 0.01%. As can be clearly seen in Fig. 2, the swimming time of the water-immersed breeding group control group is meaningfully shortened compared with the general breeding group. When the arachidonic acid in the test sample was ingested, the shortening inhibition effect of the swimming time was observed, and it was confirmed that the anti-fatigue effect was obtained even if l摄取mg/kg was ingested. Example 3 (Formulation Example 1: Preparation of Capsule) -17- 200930358 100 parts by mass of gelatin and 35 parts by mass of food additive were added with glycerin, and dissolved at 50 to 60 ° C to prepare The gelatin capsule has a viscosity of 200 cp. Further, arachidonic acid-containing fat (SUNTGA (registered trademark) 40S) was mixed with 0.05% by mass of vitamin E oil to prepare a content. The gelatin film and the contents were subjected to capsule formation and drying in accordance with a usual method to prepare soft capsules each containing 180 mg of the contents. ❹ (Formulation Example 2: Preparation of fat infusion solution) 400 g of arachidonic acid-containing fat (SUNTGA (registered trademark) 40S), 48 g of purified lecithin, 20 g of oleic acid, 10 g of glycerin and 0.1 ml of 40 ml were added. After the caustic soda of N was dispersed by a homomixer, distilled water for injection was added to 4 liters. The emulsion was further emulsified using a high-pressure spray emulsifier to prepare a lipid emulsion. Then, the lipid emulsion was dispensed into a plastic packaging bag every 200 ml, and then subjected to high pressure steam sterilization treatment at 121 ° C for 20 minutes to prepare a fat infusion solution. (Formulation Example 3: Preparation of fruit juice) 2 g of β-cyclodextrin was added to 20 ml of 20% aqueous ethanol solution, and while stirring with a stirrer, 1 〇〇mg of arachidonic acid-containing fat ( SUNTGA (registered trademark) was added. 40S is mixed with 0.05% by mass of vitamin E) and incubated at 50 ° C for 2 hours. After cooling to room temperature (about 1 hour), stirring was continued while 10 hours of incubation at 4 ° C. The resulting precipitate was recovered by centrifugation, washed with n-hexane, and then freeze-dried by -18-200930358 to obtain l-8 g of a cyclodextrin cage compound containing arachidonic acid-containing triglyceride. The lg powder was uniformly mixed with 500 ml of juice to prepare a juice containing triglyceride containing arachidonic acid. BRIEF DESCRIPTION OF THE DRAWINGS [Fig. 1] is a graph showing the anti-fatigue action of arachidonic acid in the first embodiment. Fig. 2 is a graph showing the anti-fatigue action of arachidonic acid in the second embodiment.
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