TWI589329B - 感覺改善劑 - Google Patents
感覺改善劑 Download PDFInfo
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- TWI589329B TWI589329B TW101136546A TW101136546A TWI589329B TW I589329 B TWI589329 B TW I589329B TW 101136546 A TW101136546 A TW 101136546A TW 101136546 A TW101136546 A TW 101136546A TW I589329 B TWI589329 B TW I589329B
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Classifications
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Description
本發明係關於以下列乳來源蛋白質及/或乳來源蛋白質之分解物作為有效成分之具有改善末梢神經鈍化的效果的感覺改善劑、及摻合有該感覺改善劑之感覺改善用飲食品或飼料、化妝品。
近年來,伴隨高齡化,骨質粗鬆症或認知症等由於年老所致的疾病的增加成為社會問題。所以,為了預防或改善如此的伴隨年老的疾病,已開發出了各式各樣的醫藥品。但是由於醫藥品總是需要考慮副作用,所以近年來已有人嘗試改變食生活、或利用攝取特定的食品成分而預防或改善伴隨年老的疾病。例如:對於骨質粗鬆症,已知藉由攝取牛乳所含之鹼性蛋白質來預防或改善該疾病。又,已知有以牛乳中含較多量的磷脂質即神經鞘磷脂(sphingomyelin)作為有效成分之預防或改善阿茲海默型記憶障礙的記憶障礙預防治療劑。
伴隨年老的症狀之一可列舉末梢感覺鈍化,但是該末梢的感覺鈍化,不只是因為年老,在糖尿病等疾病也會發生。末梢感覺的鈍化,會發生例如碰觸到熱的物品時無法正確地感覺到熱,燒傷等危險性增高,或是由於痛覺鈍化造成較晚發現受傷此類的問題。近年來,為了避免如此的危險性,針對改善由於年老或疾病造成之末梢感覺鈍化的研究正在進展。例如據報告:有增加外因性神經醯胺(ceramide)或內因性神經醯胺之生合成的效果的
酵素即神經鞘磷脂酶或磷脂醯膽鹼專一性磷脂酶C,會經由株化纖維母細胞3T3細胞,即由於3T3細胞所分泌之神經營養因子而促進神經模式細胞P-12細胞的形態分化(非專利文獻1)。神經模式細胞之形態分化的促進,啟示於末梢之感覺鈍化之改善效果。但由於上述神經醯胺或酵素並非食品成分,所以在使用時必須探討安全性。在如此的狀況,希望能有更安全且藉由日常攝取或塗佈於皮膚而有改善末梢感覺鈍化之效果的劑。
乳所含成分已知有各式各樣的生理活性,但對於末梢感覺的鈍化,已知乳來源的神經鞘磷脂或磷脂質有改善的效果(專利文獻1及專利文獻2)。但是針對乳來源蛋白質對於末梢感覺之鈍化的效果尚為未知。乳來源蛋白質,例如:乳過氧化酶(lactoperoxidase)、乳鐵蛋白、胱蛋白(cystatin)、血管生成素(angiogenin)。
乳過氧化酶係在乳中存在且含有血質鐵的糖蛋白質,其詳細結構至今尚不明瞭。針對乳過氧化酶的利用,已發現於生體內有過氧化脂質之生成抑制效果,且已知作為防止視力下降、運動能力下降、免疫機能下降等抗老化劑或作為肝機能改善劑,又,由於有低齲齒作用故已知利用於作為低齲齒營養組成物。但是針對乳過氧化酶及其以蛋白質分解酵素分解獲得之酵素分解物具有改善末梢神經之鈍化的作用且作為感覺改善劑為有用係為未知。
針對乳鐵蛋白及其分解物,已知有防止病原菌附著於細胞的作用、抗病毒作用等。又,據報告:藉由與上皮細胞成長因子混合,會提高上皮細胞成長因子單獨的皮膚細胞賦活作用。又,一般而言,乳鐵蛋白已知有減輕伴隨疼痛的緊張或精神性緊張的作用。但是針對乳鐵蛋白及其以蛋白質分解酵素分解獲得之酵素分解物具有改善末梢神經之鈍化的作用且作為感覺改善劑有用尚為未知。
胱蛋白,係作為半胱胺酸蛋白酶抑制劑,抑制活性中心具有SH基之半胱胺酸蛋白之蛋白質分解活性之物質,在動物組織、細胞、血液及尿中可
找到。又,已確認病毒增殖抑制作用為胱蛋白的有用作用。但是針對胱蛋白及其以蛋白質分解酵素分解而得之酵素分解物具有改善末梢神經鈍化之作用且作為感覺改善劑為有用尚為未知。
血管生成素為一種血管形成因子。人的血管生成素已知為分子量14,400的蛋白質,在血液或乳中存在。針對牛血管生成素已從牛乳中之血管生成素予以單離並已有人報告胺基酸序列的定序。又,針對從牛乳製造血管生成素,已有人揭示使牛乳吸附於陽離子交換層析後,以弱有機酸之鹼金屬鹽水溶液溶出,並將該溶出液再度以陽離子交換層析與凝膠過濾層析予以回收的方法。再者,由於發現血管生成素會專一性地抑制黑色素瘤細胞B-16細胞的黑色素產生,故據報告也可作為美白劑。但是針對血管生成素及其以蛋白質分解酵素分解而獲得之酵素分解物具有改善末梢神經鈍化之作用且作為感覺改善劑有用尚為未知。
專利文獻1:日本特開2009-126787號公報
專利文獻2:日本特開2009-126788號公報
非專利文獻1:Cosmetology研究報告(2002)第10號
本發明之課題為提供一種感覺改善劑,其係安全且可藉由日常攝取或塗佈於皮膚而顯示改善末梢感覺鈍化的效果。又,本發明之課題為提供藉由經口攝取或塗佈於皮膚而顯示於末梢感覺之鈍化改善效果之感覺改善用飲食品或飼料、化妝品。
本案發明人等有鑑於該等問題,針對安全且對於感覺鈍化顯示優異改善效果之成分努力尋找,結果發現將乳來源蛋白質及/或乳來源蛋白質分解物任一者經口攝取、或直接塗佈在皮膚,能改善感覺鈍化,尤其末梢感覺的鈍化。而,使用該等作為有效成分,完成了感覺改善劑。又,發現:可將該感覺改善劑摻合於飲食品或飼料等以製成感覺改善用飲食品或飼料、化妝品,乃完成本發明。又,本說明書係以含於乳中作為特徵,將乳過氧化酶、乳鐵蛋白、胱蛋白、血管生成素記載為「乳來源蛋白質」,但是本發明之感覺改善劑使用之該等蛋白質不一定要為乳來源,也可為人工合成者或從血液等精製者。
亦即本發明,係
(1)一種感覺改善劑,其係以乳來源蛋白質及/或乳來源蛋白質分解物作為有效成分。
(2)如(1)之感覺改善劑,其中,前述乳來源蛋白質係選自於乳過氧化酶、乳鐵蛋白、胱蛋白、血管生成素中之任一種以上。
(3)如(1)至(2)之感覺改善劑,其中,前述乳來源蛋白質分解物係將乳來源蛋白質以蛋白質分解酵素分解而得者。
(4)如(3)之感覺改善劑,其中,前述蛋白質分解酵素係選自於由胃液素、胰蛋白酶、胰凝乳蛋白酶、胰酶構成之群組中之一種以上。
(5)一種感覺改善用飲食品或飼料、化妝品,其係摻合有如(1)至(4)中任一項之成分。
(6)一種改善哺乳動物之感覺之方法,其係利用使哺乳動物攝取乳來源蛋白質及/或乳來源蛋白質分解物或塗佈於皮膚。
(7)如(6)之方法,其中,該哺乳動物為人且係使成人每人每日攝取10mg以上的乳來源蛋白質及/或乳來源蛋白質分解物。
本發明之感覺改善劑可獲得改善末梢感覺鈍化的效果。
本發明之特徵在於:以乳來源蛋白質及/或乳來源蛋白質分解物作為有效成分。乳來源蛋白質可列舉乳過氧化酶、乳鐵蛋白、胱蛋白、血管生成素,可使用從人、牛、水牛、山羊、綿羊等哺乳類之乳汁獲得者或以基因工程的方法生產者。又,乳來源蛋白質分解物,係能使蛋白質分解酵素作用於該等乳來源蛋白質而能得到者。
乳過氧化酶係從哺乳動物的乳汁製備。供給源可列舉人、牛、水牛、山羊、綿羊等的乳汁。乳過氧化酶為公知物質,也有市售者,為了製造可利用公知方法,使用例如在工業上利用碸化擔體將乳過氧化酶精製之方法(日本特開平3-109400號公報)。又,本發明中,也可使用以基因工程方法生產的乳過氧化酶。乳過氧化酶分解物,係將上述乳過氧化酶利用胰蛋白酶、胰酶、胰凝乳蛋白酶、胃液素、木瓜蛋白酶、血管舒緩素、組織蛋白酶、嗜熱菌蛋白酶、V8蛋白酶等蛋白質分解酵素進行限定分解成分子量10,000以下而得的胜肽混合物。分子量下限為500以上為較佳。
乳鐵蛋白係從哺乳動物的乳汁製備。供給源可列舉人、牛、水牛、山羊、綿羊等的乳汁。乳鐵蛋白為公知物質,也有市售者,製造時可以利用公知方法,例如工業化利用使用碸化擔體精製乳鐵蛋白之方法(日本特開平3-109400號公報)。又,本發明中,也可使用利用基因工程的方法生產的乳鐵蛋白。乳鐵蛋白分解物,為將上述乳鐵蛋白利用胰蛋白酶、胰酶、胰凝乳蛋白酶、胃液素、木瓜蛋白酶、血管舒緩素、組織蛋白酶、嗜熱菌蛋白酶、V8蛋白酶等蛋白質分解酵素進行限定分解使分子量為10,000以下而得之胜肽混合物。分子量之下限為500以上為較佳。
胱蛋白,可使用包含人、牛、水牛、山羊、綿羊等的乳汁來源的各種來源者。例如:人及牛來源的胱蛋白的基因序列已明瞭,可利用基因重組生產,但是本發明中,也可使用以基因工程的方法生產的胱蛋白。又,胱蛋白在牛初乳中含有較大量,也可從乳汁回收。再者,胱蛋白也可從細胞培養的培養液回收,也可使用如此之細胞來源者。例如針對乳來源的胱蛋白,也可依公知方法(日本特開2000-281587號公報等)製造,可從生乳或奶粉、脫脂乳、還原乳等進行加熱處理、加鹽處理、乙酶處理、離子交換層析或凝膠過濾層析等各種層析處理、超過濾處理等而獲得胱蛋白。胱蛋白分解物,可使用將胱蛋白以胰蛋白酶、胰酶、胰凝乳蛋白酶、胃液素、木瓜蛋白酶、血管舒緩素(kallikrein)、組織蛋白酶(cathepsin)、嗜熱菌蛋白酶(thermolysin)、V8蛋白酶等蛋白質分解酵素進行限定分解使分子量為8,000以下而得之胜肽混合物。惟分子量之下限為500以上較佳。
就血管生成素之供給源而言,由於人、牛、水牛、山羊、綿羊等在分娩後1~7日以內,尤佳為分娩後1~5日以內的初乳的血管生成素含量高故為理想,但也可使用通常泌乳期的乳汁作為本發明原料。為了製造可使用公知方法,例如工業化利用組合陽離子交換層析與凝膠過濾層析而精製血管生成素之方法(日本特開平2-296000號公報等)。血管生成素分解物,為將上述血管生成素以胰蛋白酶、胰酶、胰凝乳蛋白酶、胃液素、木瓜蛋白酶、血管舒緩素、組織蛋白酶、嗜熱菌蛋白酶、V8蛋白酶等蛋白質分解酵素進行限定分解使分子量為10,000以下而得之胜肽混合物。分子量之下限為500以上為較佳。
作為本發明之感覺改善劑,可以直接使用上述乳來源蛋白質,尤其乳過氧化酶或乳鐵蛋白、胱蛋白、血管生成素、或使蛋白質分解酵素作用於乳來源蛋白質而得之分解物作為感覺改善劑,但此外,也可與糖類或脂質、蛋白質、維生素類、礦物質類、風味劑等其他在醫藥品、飲食品或飼料通常使用的原材料等混合、或進一步依常法製劑為粉末劑、顆粒劑、錠劑、膠囊劑、飲用劑等。又,塗佈劑可使用乳液、乳霜、塗劑(lotion)、面膜等通常的塗佈形態。該等塗佈劑可依常法製造並在其製造過程適當摻合為本
發明有效成分之乳來源蛋白質及/或乳來源蛋白質分解物,也可作為化妝品使用。又,乳來源蛋白質及/或乳來源蛋白質分解物以外,也可同時使用其他顯示感覺改善效果的成分,例如神經醯胺或神經鞘磷脂酶、神經鞘磷脂等。本發明之感覺改善劑之有效量,於後述小鼠的試驗,藉由體重每1kg經口攝取乳來源蛋白質及/或乳來源蛋白質分解物10mg以上,較佳為20mg以上,會改善末梢感覺,所以通常成人每人每日利用攝取乳來源蛋白質及/或乳來源蛋白質分解物10mg以上,較佳為20mg以上,可期待改善改覺,尤其末梢感覺鈍化,所以能確保該必要量即可。又,塗佈於皮膚的情形,以皮膚塗佈劑的總量作為基準,為0.001~40重量%,更佳為0.1~10重量%。
作為本發明之感覺改善用飲食品,可在通常的飲食品,例如優格、乳飲料、威化餅、甜點等中摻合乳來源蛋白質及/或乳來源蛋白質分解物即可。針對該等感覺改善用飲食品,為了使成人每人每日攝取乳來源蛋白質及/或乳來源蛋白質分解物10mg以上,雖取決於飲食品形態,但宜飲食品每100g摻合乳來源蛋白質及/或乳來源蛋白質分解物0.5~2000mg較佳。又,作為本發明之感覺改善用飼料,可於通常飼料,例如家畜用飼料或寵物食品等中摻合乳來源蛋白質及/或乳來源蛋白質分解物即可。例如:針對該等飼料當摻合乳來源蛋白質及/或乳來源蛋白質分解物的情形,為了攝取乳來源蛋白質及/或乳來源蛋白質分解物10mg以上,飼料每100g宜摻合乳來源蛋白質及/或乳來源蛋白質分解物0.5~2000mg較佳。
本發明中,摻合乳來源蛋白質及/或乳來源蛋白質分解物的方法不特別限制,例如:為了添加、摻合於溶液中,將乳來源蛋白質及/或乳來源蛋白質分解物懸浮或溶解於去離子水並攪拌混合後,製備為醫藥品、飲食品或飼料的形態使用。攪拌混合的條件,只要能使乳來源蛋白質及/或乳來源蛋白質分解物均勻混合即可,也可使用超分散器(ultradisperser)或TK均質混合機等並攪拌混合。又,該組成物的溶液,為了容易使用在醫藥品、飲食品或飼料,也可視需要以RO膜等濃縮或冷凍乾燥後使用。本發明中,可進行醫藥品、飲食品或飼料之製造時通常使用之殺菌處理,為粉末狀時也可乾熱殺菌。因此本發明可製造含有乳來源蛋白質及/或乳來源蛋白質分解物之
液狀、凝膠狀、粉末狀、顆粒狀等各種形態之醫藥品、飲食品或飼料。
以下舉實施例及試驗例針對本發明更詳細說明,但該等僅僅是單純例示而已,本發明不限定於該等。
將填充有為陽離子交換樹脂之碸化Chitopearl(富士紡績公司製)400g的管柱(直徑5cm×高度30cm)以去離子水充分洗滌後,於該管柱以流速25ml/min通入未殺菌脫脂乳40L(pH6.7)。通液後將管柱以去離子水充分洗滌,以含2.0M氯化鈉之0.02M碳酸緩衝液(pH7.0)進行溶出。並使含有乳過氧化酶之溶出級分吸附於S-SepharoseFF管柱(GE Healthcare公司製),以去離子水充分洗滌,並以10mM磷酸緩衝液(pH7.0)平衡化之後,以0~2.0M氯化鈉的線性梯度將已吸附的級分溶出,並回收含有乳過氧化酶之級分。並且將該級分以使用HiLoad16/60Superdex75pg(GE Healthcare公司製)之凝膠過濾層析處理,獲得乳過氧化酶3.0g。又,以如此的方式獲得之乳過氧化酶之純度為94%,可直接作為感覺改善劑(實施例品1)使用。
將實施例1獲得之乳過氧化酶1g溶於水200ml,加入為蛋白質分解酵素之胰酶(Sigma公司製)使得最終濃度為0.01重量%,於37℃進行5小時酵素處理。然後,於90℃進行5分鐘加熱處理使酵素失活後,冷凍乾燥獲得乳過氧化酶分解物0.8g。又,以如此方式獲得之乳過氧化酶分解物之分子量為10,000以下,可直接作為感覺改善劑(實施例品2)使用。
將實施例1獲得之乳過氧化酶1g溶於水200ml,加入為蛋白質分解酵素之胰蛋白酶(Sigma公司製)使得最終濃度為0.01重量%,於37℃進行5小時酵素處理。然後,於90℃進行5分鐘加熱處理使酵素失活後,冷凍乾燥獲得乳過氧化酶分解物0.9g。又,以該方式獲得之乳過氧化酶分解物之分子量為10,000以下,可直接作為感覺改善劑(實施例品3)使用。
將填充有為陽離子交換樹脂之碸化Chitopearl(富士紡績公司製)400g之管柱(直徑5cm×高度30cm)以去離子水充分洗滌後,於該管柱以流速25ml/min通入未殺菌脫脂乳40L(pH6.7)。通液後將管柱以去離子水充分洗滌,並以含2.0M氯化鈉之0.02M碳酸緩衝液(pH7.0)進行溶出。然後使含乳鐵蛋白之溶出級分吸附於S-Sepharose FF管柱(GE Healthcare公司製),以去離子水充分洗滌,並以10mM磷酸緩衝液(pH7.0)平衡化後,以0~2.0M氯化鈉的線性梯度將已吸附的級分溶出,回收含乳鐵蛋白的級分。然後將該級分以使用HiLoad 16/60 Superdex75 pg(GE Healthcare公司製)之凝膠過濾層析處理,獲得乳鐵蛋白8.0g。又,以該方式獲得之乳鐵蛋白之純度為96%,可直接作為感覺改善劑(實施例品4)使用。
將實施例4獲得之乳鐵蛋白1g溶於水200ml,加入為蛋白質分解酵素之胰酶(Sigma公司製)使最終濃度為0.01重量%,於37℃進行5小時酵素處理。然後,於90℃進行5分鐘加熱處理使酵素失活後冷凍乾燥,獲得乳鐵蛋白分解物0.8g。又,以此方式獲得之乳鐵蛋白分解物之分子量為10,000以下,可以直接作為感覺改善劑(實施例品5)使用。
將實施例4獲得之乳鐵蛋白1g懸浮於水200ml,加入為蛋白質分解酵素之胰蛋白酶(Sigma公司製)使最終濃度為0.01重量%,於37℃進行5小時酵素處理。然後,於90℃進行5分鐘加熱處理使酵素失活後冷凍乾燥,獲得乳鐵蛋白分解物0.9g。又,以此方式獲得之乳鐵蛋白分解物之分子量為10,000以下,可直接作為感覺改善劑(實施例品6)使用。
將填充有S Sepharose3,000g之管柱以去離子水充分洗滌,通入脫脂乳10,000L,並以去離子水充分洗滌後,以0.1~1.0M之氯化鈉之直線濃度梯度
進行溶出。將獲得之級分於90℃進行10分鐘加熱處理,並離心分離以去除沈澱。然後將含牛乳來源鹼性胱蛋白之溶出級分再度以MonoS離子交換層析區分。再者,將該級分利用FPLC系統進行MonoQ離子交換層析、Superose12凝膠過濾層析,再以HPLC系統依序進行羥基磷灰石層析及C4逆相層析處理,獲得胱蛋白58mg(級分A)。又,以該方式獲得之胱蛋白可直接作為感覺改善劑(實施例品7)使用。
將5%乳清蛋白質溶液10,000L於90℃進行10分鐘加熱處理,並以離心分離去除沈澱。再者,於管柱中填充使羧基甲基化木瓜蛋白酶鍵結於Tresyl-Toyopearl(Tresyl-Toyopearl、東曹公司製)而得之擔體,之後以0.5M氯化鈉溶液平衡化,將先前的乳清蛋白質溶液通入。通液後,以0.5M氯化鈉溶液及含0.1%Tween20之0.5M氯化鈉溶液依序洗滌管柱。其次,以20mM乙酸-0.5M氯化鈉溶液使半胱胺酸蛋白酶溶出。將溶出級分立即以1M氫氧化鈉溶液中和,進行MonoS陰離子交換層析,並以HPLC系統依序進行羥基磷灰石層析及C4逆相層析處理予以區分,獲得牛乳來源鹼性胱蛋白48mg(級分B)。又,該方式獲得之胱蛋白可直接作為感覺改善劑(實施例品8)使用。
將實施例7獲得之級分A 25mg懸浮於水100ml,並加入胰酶使最終濃度成為1%,於37℃進行5小時酵素處理。然後於90℃進行5分鐘加熱處理使酵素失活後冷凍乾燥,獲得胱蛋白分解物23mg(級分C)。又,實施例8獲得之級分B25mg同樣進行處理,獲得胱蛋白分解物24mg(級分D)。又,以此方式獲得之胱蛋白分解物之分子量為8,000以下,可直接作為感覺改善劑(實施例品9)使用。
將填充有為陽離子交換樹脂之碸化Chitopearl(富士紡績公司製)400g之管柱(直徑5cm×高度30cm)以去離子水充分洗滌後,於該管柱以流速
25ml/min通入未殺菌脫脂乳40L(pH6.7)。通液後將管柱以去離子水充分洗滌,以含2.0M氯化鈉之0.02M碳酸緩衝液(pH7.0)進行溶出。然後,使含血管生成素之溶出級分吸附於S-Sepharose FF管柱(GE Healthcare公司製),以去離子水充分洗滌,以10mM磷酸緩衝液(pH7.0)平衡化後,以0~2.0M氯化鈉之線性梯度使已吸附的級分溶出,回收含血管生成素之級分。然後,將該級分以使用HiLoad16/60Superdex75 pg(GE Healthcare公司製)之凝膠過濾層析處理,獲得含多量血管生成素之級分1.8g。又,以此方式獲得之含多量血管生成素之級分中之血管生成素含量為10%,可直接作為感覺改善劑(實施例品10)使用。
將填充有為陽離子交換樹脂之碸化Chitopearl(富士紡績公司製)3,000g之管柱以去離子水充分洗滌後,於該管柱通入未殺菌脫脂乳100L(pH6.7)。其次將該管柱以去離子水充分洗滌後,以0.1~2.0M氯化鈉的直線濃度梯度進行溶出。然後將含有血管生成素之溶出級分以S-Sepharose陽離子交換層析(GE Healthcare公司製)區分,將獲得之含血管生成素之級分以90℃進行10分鐘加熱處理,並離心分離以去除沈澱。再者,將該含血管生成素之級分以MonoS陽離子交換層析、Superose12凝膠濾過層析、羥基磷灰石層析及C4逆相層析依序處理,獲得血管生成素55mg。又,以該方式獲得之血管生成素之純度為99%,可直接作為感覺改善劑(實施例品11)使用。
將實施例11獲得之血管生成素5mg溶於水10ml,加入胰酶(Sigma公司製)使最終濃度成為0.01重量%,於37℃進行5小時酵素處理。然後於90℃進行5分鐘加熱處理使酵素失活後冷凍乾燥,獲得血管生成素分解物4.0mg。以此方式獲得之血管生成素分解物之分子量為10,000以下,可直接作為感覺改善劑(實施例品12)使用。
將實施例11獲得之血管生成素5mg溶於水10ml,加入胰蛋白酶(Sigma
公司製)使最終濃度成為0.01重量%,於37℃進行5小時酵素處理。然後,於90℃進行5分鐘加熱處理使酵素失活後冷凍乾燥,獲得血管生成素分解物4.2mg。以此方式獲得之血管生成素分解物之分子量為10,000以下,可直接作為感覺改善劑(實施例品13)使用。
將已知存在於皮膚之纖維母細胞的細胞株即3T3細胞以添加有濃度0.03~1%之實施例品1及實施例品3、實施例品4、實施例品5、實施例品7、實施例品9之級分C、實施例品11、實施例品13的狀態培養2天。又,作為對照,不添加實施例品,將3T3細胞培養2天(對照)。使用該等培養上清來培養神經之模式細胞PC-12細胞,觀察當3T3細胞分泌神經營養因子的情形的PC-12細胞的形態分化。
其結果,有添加實施例品時之培養上清,所有PC-12細胞皆強分化。又,重複該實驗數次,並於光學顯微鏡下觀察分化之比例,結果於有添加實施例品的情形,認為有95%以上的細胞有分化。另一方面,對照的培養上清,未使PC-12細胞分化,在數次重複實驗中,於光學顯微鏡下之觀察完全未認為有分化。由此可知乳來源蛋白質及/或乳來源蛋白質分解物促進從3T3細胞分泌神經營養因子,並促進神經模式細胞PC-12細胞之分化。
以Woolfe、MacDonald等人開發的對熱刺激之行動學研究法即熱板試驗,以熱刺激評價感覺改善效果。將24週大的無毛小鼠(Hos:HR-1)各6隻為1組,分成13組。將實施例品2及實施例品6、實施例品8、實施例品9之級分D、實施例品10、實施例品12,以小鼠體重每1kg各10mg、20mg的量,1日1回以探管經口投予,或將不含任一實施例品僅含溶劑的試液以1日1次以探管經口投予(對照;0mg),飼養4週。投予結束時將小鼠放置在54℃的熱板,測定小鼠的腳出現從熱板離開、站起、跳躍等逃避
行動的時間。熱刺激的最大強度設為30秒,此時之值定為30秒。其結果如表1。
如表1所示,藉由攝取10mg的實施例品2及實施例品6、實施例品8、實施例品9之級分D、實施例品10、實施例品12,可認為逃避行動陽性反應時間有縮短的傾向,於攝取20mg則顯著縮短。由此可知,藉由攝取實施例品2及實施例品6、實施例品8、實施例品9之級分D、實施例品10、實施例品12,能預防、改善感覺,尤其末稍感覺的鈍化。
將感到手感覺鈍化的健康年長者(平均年齡75±3歳)各10名為1組,分成9組。各設為:任一實施例品都未攝取的組(A組)、攝取10mg實施例品1之組(B組)、攝取20mg之組(C組)、攝取10mg實施例品4之組(D組)、攝取20mg之組(E組)、攝取10mg實施例品7之組(F組)、攝取20mg之組(G
組)、攝取10mg實施例品11之組(H組)、攝取20mg之組(I組),使攝取6週。攝取前及6週的攝取結束後,使用為表面知覺計之痛覺計(algesimeter)(Inter-cross公司製),以上腕內側的痛覺作為基準,依使用方法測定手掌與足底足弓的痛覺,以正常、下降I~III的共4級評價。其結果如表2、表3。又,又6週攝取結束後,對各年長者針對手感覺的改善實施意見調査。其結果如表4、表5。
以5種粗胖度不同的針及5個支點位置評價痛覺。首先用最細的1pin在上腕內側轉動,向受測者確認正常痛覺的程度。其次,於手掌與腳底以1pin,握持支座將支點的位置依序改變並轉動,找尋感到與最初感到的痛覺為同程度痛楚的支點的位置。
痛覺計,係設計為當於上腕內側轉動1pin(支點50g)時,於手掌會產生與2pin(50g)為相同痛楚之用,依使用方法以下列方式評價。又,於痛楚的評價計分,並計算平均值。
若與2pin(50g)時為相同痛楚,則為正常(0分)
若與1pin(50g)時為相同痛處,則為下降I(1分)
若與1pin(60g)時為相同痛楚,則為下降II(2分)
若與1pin(70g)時為相同痛楚,則為下降III(3分)
如表2至表5所示,藉由攝取10mg實施例品1或實施例品4、實施例品7、實施例品11,認為手、腳底的感覺有改善的傾向,攝取20mg則明顯改善。通常,成人每人每日藉由攝取乳來源蛋白質及/或乳來源蛋白質分解物10mg以上,較佳為20mg以上可期待改善感覺,尤其末梢感覺的鈍化。
使用實施例3獲得之乳過氧化酶分解物(實施例品3),以表6記載的比例將原料混合,製造感覺改善用化妝品(乳霜)。
將感到手感覺鈍化的健康年長者(平均年齡75±3歳)各15名為1組,分成A組、B組兩組。A組,1日1次在手跟腳全體塗佈與實施例品14以同樣方法製造之不含感覺改善劑之化妝品(乳霜),B組1日1次在手跟腳全體塗佈實施例品14的感覺改善用化妝品(乳霜)。塗佈期間設為6週,於塗佈前與6週的塗佈結束後,使用表面知覺計痛覺計(Inter-cross(股)公司製),以上腕內側的痛覺作為基準,將手掌與腳底足弓的痛覺依使用方法,以正常、下降I~III的共計4級測定。其結果如表7、表8。又,6週的塗佈結束後,對於各受測者針對手的感覺改善進行意見調査。其結果如表9、表10。又,針對測定方法與試驗例3以同樣方式進行。
如表7至表10所示,藉由塗佈實施例品14之感覺改善用化妝品(乳霜),認為手、腳底的感覺有改善的傾向。由此可知藉由塗佈含有本發明之感覺改善劑的乳霜,能期待感覺鈍化尤其末梢感覺鈍化改善。
將實施例品5之乳鐵蛋白分解物5g溶於4995g的去離子水,以TK均質混合機(TKROBOMICS;特殊機化工業公司製)以6000rpm攪拌混合30分鐘,獲得乳鐵蛋白分解物100mg/100g的乳鐵蛋白分解物溶液。於該乳鐵蛋白分解物溶液5.0kg中,摻合酪蛋白4.0kg、黃豆蛋白質5.0kg、魚油1.0kg、紫蘇油3.0kg、糊精18.0kg、礦物質混合物6.0kg、維生素混合物1.95kg、乳化劑2.0kg、安定劑4.0kg、香料0.05kg,填充於200ml的加壓加熱軟袋,以加壓加熱殺菌機(第1種壓力容器、TYPE:RCS-4CRTGN、日阪製作所公司製)於121℃進行20分鐘殺菌,製造本發明之感覺改善用液狀營養組成物50kg。如此獲得之感覺改善用液狀營養組成物中完全沒有沉澱等且風味未感到異常。又,該感覺改善用液狀營養組成物中,每100g含有乳鐵蛋白分解物10mg。
將實施例品8的胱蛋白2g溶於708g的去離子水,以超分散器(ULTRA-TURRAXT-25;IKA Japan公司製)以9500rpm攪拌混合30分鐘。於該溶液中添加山梨醇40g、酸味料2g、香料2g、果膠5g、乳清蛋白質濃縮物5g、乳酸鈣1g、去離子水235g,攪拌混合後填充於200ml的附蓋軟袋,於85℃殺菌20分鐘後蓋緊,製備本發明的感覺改善用凝膠狀食品5袋(內容量200g)。以此方式獲得之感覺改善用凝膠狀食品完全無沉澱等且風味未感到異常。又,該感覺改善用凝膠狀食品中,每100g含有胱蛋白200mg。
將酸味料2g溶於706g去離子水後,溶解實施例品11的血管生成素4g,以超分散器(ULTRA-TURRAXT-25;IKA Japan公司製)以9500rpm攪拌混合30分鐘。添加麥芽糖醇(maltitol)100g、還原糖漿20g、香料2g、去離子水166g後,填充於100ml玻璃瓶,於95℃殺菌15秒後蓋緊,製備感覺改善用飲料10瓶(內容量100ml)。以此方式獲得之感覺改善用飲料,完全無
沉澱,也未感到風味異常。又,該感覺改善用飲料中,每100g含有血管生成素400mg。
將實施例品2之乳過氧化酶分解物2kg溶於98kg去離子水,以TK均質混合機(MARKII 160型;特殊機化工業公司製)以3600rpm攪拌混合40分鐘,獲得含有乳過氧化酶分解物2g/100g之乳過氧化酶分解物溶液。於該乳過氧化酶分解物溶液10kg摻合大豆粕12kg、脫脂粉乳14kg、大豆油4kg、玉米油2kg、棕櫚油23.2kg、玉米澱粉14kg、麵粉9kg、麩2kg、維生素混合物5kg、纖維素2.8kg、礦物質混合物2kg,於120℃殺菌4分鐘,製造本發明之感覺改善用犬飼育飼料100kg。又,該感覺改善用犬飼育飼料中,每100g含有乳過氧化酶分解物200mg。
以表11所示之摻合將原料混合後,依常法成型、打錠為1g,製造本發明之感覺改善劑。又,該感覺改善劑1g中含有乳過氧化酶100mg。
以表12記載的比例將原料混合,製造感覺改善用化妝品(塗劑)。
Claims (2)
- 一種乳來源蛋白質及/或乳來源蛋白質分解物的用途,係於製造用於改善哺乳動物之感覺之感覺改善劑時使用選自於分子量500以上10,000以下的乳過氧化醇分解物、分子量500以上10,000以下的乳鐵蛋白分解物、分子量500以上8,000以下的胱蛋白、分子量500以上10,000以下的血管生成素中之任一種以上之乳來源蛋白質及/或乳來源蛋白質分解物,且係利用使哺乳動物攝取乳來源蛋白質及/或乳來源蛋白質分解物或塗佈於皮膚。
- 如申請專利範圍第1項之乳來源蛋白質及/或乳來源蛋白質分解物的用途,其中,哺乳動物為人,且係使成人每人每日攝取乳來源蛋白質及/或乳來源蛋白質分解物10mg以上。
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JP2011020973A (ja) * | 2009-07-17 | 2011-02-03 | Nrl Pharma Inc | 難治性の皮膚潰瘍治療用の医薬組成物、及び、皮膚潰瘍予防用化粧料 |
JP5955499B2 (ja) | 2010-09-30 | 2016-07-20 | 雪印メグミルク株式会社 | 皮膚コラーゲン産生促進剤 |
JP2012077018A (ja) | 2010-09-30 | 2012-04-19 | Snow Brand Milk Products Co Ltd | 皮膚コラーゲン産生促進剤 |
AU2012210013C1 (en) * | 2011-01-26 | 2017-06-01 | Megmilk Snow Brand Co., Ltd. | Sense-improving agent |
JP5890100B2 (ja) | 2011-02-09 | 2016-03-22 | 雪印メグミルク株式会社 | 皮膚コラーゲン産生促進剤 |
JP2012188384A (ja) | 2011-03-10 | 2012-10-04 | Snow Brand Milk Products Co Ltd | 美肌剤 |
JP2013079216A (ja) | 2011-10-04 | 2013-05-02 | Snow Brand Milk Products Co Ltd | 感覚改善剤 |
-
2011
- 2011-10-04 JP JP2011220444A patent/JP2013079216A/ja active Pending
-
2012
- 2012-10-02 AU AU2012319579A patent/AU2012319579B2/en not_active Ceased
- 2012-10-02 US US14/349,456 patent/US20140249094A1/en not_active Abandoned
- 2012-10-02 EP EP12838671.1A patent/EP2764870A4/en not_active Withdrawn
- 2012-10-02 KR KR1020147011002A patent/KR20140072893A/ko not_active Application Discontinuation
- 2012-10-02 WO PCT/JP2012/075544 patent/WO2013051572A1/ja active Application Filing
- 2012-10-02 CA CA2849754A patent/CA2849754A1/en not_active Abandoned
- 2012-10-03 TW TW101136546A patent/TWI589329B/zh not_active IP Right Cessation
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2015
- 2015-10-02 US US14/873,622 patent/US10016481B2/en not_active Expired - Fee Related
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2017
- 2017-07-19 AU AU2017206200A patent/AU2017206200A1/en not_active Abandoned
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CA2849754A1 (en) | 2013-04-11 |
US10016481B2 (en) | 2018-07-10 |
KR20140072893A (ko) | 2014-06-13 |
EP2764870A4 (en) | 2015-07-29 |
US20140249094A1 (en) | 2014-09-04 |
EP2764870A1 (en) | 2014-08-13 |
US20160022771A1 (en) | 2016-01-28 |
TW201318645A (zh) | 2013-05-16 |
AU2012319579B2 (en) | 2017-08-10 |
AU2012319579A1 (en) | 2014-04-17 |
JP2013079216A (ja) | 2013-05-02 |
AU2017206200A1 (en) | 2017-08-03 |
NZ623006A (en) | 2016-07-29 |
WO2013051572A1 (ja) | 2013-04-11 |
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