CN115340990A - 精氨酸酶产生促进剂 - Google Patents
精氨酸酶产生促进剂 Download PDFInfo
- Publication number
- CN115340990A CN115340990A CN202210444717.XA CN202210444717A CN115340990A CN 115340990 A CN115340990 A CN 115340990A CN 202210444717 A CN202210444717 A CN 202210444717A CN 115340990 A CN115340990 A CN 115340990A
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- Prior art keywords
- arginase
- production
- lactic acid
- present
- skin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
本发明的课题在于提供促进精氨酸酶的产生的新物质及其应用。一种精氨酸酶的产生促进剂，以乳酸菌为有效成分。
Description
技术领域
本发明涉及一种精氨酸酶产生促进剂。
背景技术
精氨酸酶是一种催化尿素生成反应的酶。精氨酸酶存在于以人为代表的哺乳类、两栖动物的肝脏、肾脏等中，在尿素循环中，将精氨酸分解而生成鸟氨酸和尿素。另外，精氨酸酶被铁、钴、锰等二价金属离子活化，被汞离子、银离子、柠檬酸等灭活。
通常，运动等后增加的血中的氨在肝脏中通过尿素合成而进行解毒(代谢)、排泄。氨的代谢过程中，由肝细胞通过尿素循环而变为尿素，由肾脏而排泄到尿中。因此，认为在因肝脏功能的降低所致的尿素循环的活性降低、肠内的氨的产生增加的情况下，血中氨浓度变为高值。因此，在先天性尿素循环酶缺乏症和肝功能异常的情况下，往往出现高氨血症。
如上所述，尿素循环主要在肝脏发挥功能，起到将氨转化为尿素的解毒功能。众所周知，该功能降低时，氨会损害肝脏细胞，引起各种肝脏疾病，并引起高氨血症等严重症状。
高氨血症的患者会出现恶心、呕吐、意识障碍、嗜睡、抽搐、呼吸障碍等症状，如果延误治疗可能会危及生命。
体内生成的氨在血中转移，在肝脏进入尿素循环，经过氨基甲酰磷酸而转化为瓜氨酸，最终由精氨酸生成尿素而解毒。作为尿素循环的酶之一的精氨酸酶是控制由精氨酸生成鸟氨酸的反应的酶。精氨酸担负氨解毒作用这样重要的功能。
作为具有精氨酸酶的活化作用的物质，已知有生药木通(AKEBIAE CAULIS)的提取物(专利文献1)、麦门冬提取物(专利文献2)、杏果汁(专利文献3)、萝藦科白前属的植物提取物(专利文献4)等植物提取物。另外，还已知利用精氨酸酶活化作用，作为奶来源的蛋白质的乳铁蛋白(专利文献5)、大豆甾醇(专利文献6)具有尿素循环活性作用、高氨血症改善作用。此外，提出了将这些提取物作为肤质改善剂使用的发明。
另外，近年来，微生物及其培养代谢产物作为功能性原料的利用受到关注。特别是已知白曲霉的菌体提取物(专利文献7)表现出精氨酸酶活化作用，此外，例示了与血压上升和血糖上升、紧密连接形成相关的基因的活化、胶原酶抑制作用、透明质酸酶抑制作用等。另外，已知乳酸菌具有免疫提高、抗炎、脂质代谢、肤质改善等多种功能。
精氨酸酶存在多个同种型。例如，已知有能够识别红细胞中的精氨酸酶和肝脏来源的精氨酸酶的抗体(专利文献8)。肝脏中的精氨酸酶被命名分类为精氨酸酶1，负责肝脏中的大部分精氨酸酶活性。肝脏中的精氨酸酶1的活性降低会延缓氨的解毒并导致高氨血症。因此，能够通过促进精氨酸酶1的活性来预防、治疗高氨血症等疾病。
另外，专利文献9公开了一种基因重组人精氨酸酶I(也称为精氨酸酶1或ARG－1)。已经阐明：精氨酸酶1以高浓度表达时，在免疫活性细胞中通过消耗精氨酸来抑制T细胞的功能而抑制过敏反应。
此外，专利文献10中发现皮肤中存在精氨酸酶1，有助于抑制伴随着紫外线照射而产生的皮肤的红斑，可以作为对紫外线的抵抗性的指标，因此提出了以精氨酸酶1为指标的晒伤的评价方法。
现有技术文献
专利文献
专利文献1：日本专利第3823373号公报
专利文献2：日本特开2003－277285号公报
专利文献3：日本特开2006－143608号公报
专利文献4：日本特开2008－255078号公报
专利文献5：日本特开2012－223100号公报
专利文献6：日本专利第6605794号公报
专利文献7：日本专利第6783962号公报
专利文献8：WO96/28732号公报
专利文献9：日本特表2010－512168号公报
专利文献10：日本专利第4446010号公报
发明内容
本发明的课题在于提供一种促进精氨酸酶的产生的新物质及其利用。
即，本发明如下所述。
〔1〕一种精氨酸酶的产生促进剂，以乳酸菌为有效成分。
〔2〕根据〔1〕所述的产生促进剂，其中，促进皮肤组织的上述精氨酸酶的产生。
〔3〕根据〔1〕或〔2〕所述的产生促进剂，其中，上述乳酸菌包含乳球菌属。
〔4〕根据〔1〕～〔3〕中任一项所述的产生促进剂，其中，将为死菌体的上述乳酸菌作为有效成分。
〔5〕一种皮肤软化剂，以乳酸菌为有效成分。
〔6〕一种肝脏组织的精氨酸酶的产生促进剂，以乳酸菌为有效成分。
〔7〕一种尿素循环活化剂，以乳酸菌为有效成分。
〔8〕一种高氨血症改善剂，以乳酸菌为有效成分。
根据本发明，能够提供促进精氨酸酶的产生的新物质及其利用。
具体实施方式
以下，对本发明的实施方式(以下，称为“本实施方式”)进行详细说明，但本发明并不限定于此，可以在不脱离其主旨的范围进行各种变形。
本实施方式的产生促进剂为以乳酸菌为有效成分的精氨酸酶的产生促进剂。另外，本实施方式还涉及该精氨酸酶的产生促进剂的利用。具体而言，如下所述。
通过使用本实施方式的产生促进剂而实现精氨酸酶的活性促进，并且实现尿素循环的活化，进而实现氨代谢的促进。因此，本实施方式的产生促进剂对高氨血症的抑制(症状的缓和)是有用的。
另外，通过使用本实施方式的产生促进剂来促进皮肤组织中的精氨酸酶的酶产生，因此皮肤组织的尿素含量增加，其结果，实现皮肤的软化，而且改善皮肤组织的保水性。此外，通过使用本实施方式的产生促进剂而实现精氨酸酶的活性促进，由此抑制皮肤组织的黑色素产生。因此，本实施方式的产生促进剂作为皮肤的美白剂也有用。也将这样的用途的药剂统一称为肌肤功能改善剂。
此外，本实施方式的产生促进剂可以适当地作为肝脏的精氨酸酶活化作用的药品、准药品、食品(功能性食品、营养补充剂、饮料等)等使用，另外，也可以适当地作为皮肤的精氨酸酶产生促进用的化妆品、药品、准药品、食品(功能性食品、营养补充剂、饮料等)等使用。将这样的用途的药剂也称为肝功能提高剂。
本实施方式中，精氨酸酶是指将精氨酸分解而生成鸟氨酸和尿素的酶，主要包含存在于肝脏中的作为同种型的精氨酸酶1。
本实施方式中使用的乳酸菌为革兰氏阳性菌，为杆菌或球菌，是相对于消耗的糖产生50％以上的乳酸的细菌。例如，可举出乳球菌属、明串珠菌属、链球菌属、片球菌属等乳酸菌。优选为乳球菌属。这些乳酸菌为一般存在于食品中的乳酸菌、食品制造中使用的乳酸菌、或者从健康人的粪便中分离出来的乳酸菌，因此不存在副作用的风险。另外，可以通过培养而容易地大量获得，因此使用培养而得到的菌体时生产成本低，比较经济。近年来，作为食品、药品或化妆品而具有多种用途。
作为通过培养而使上述的乳酸菌增殖的培养方法，可以使用以往公知的乳酸菌的培养方法，没有特别限制。例如，可举出如下方法：使用乳酸菌生长用培养基在37℃下将pH保持在中性附近而培养5～120小时、优选16～28小时，得到活菌数约107～1010/ml、优选约108～1010/ml的培养液。
本实施方式中使用的乳酸菌可以为死菌体，也可以为菌体混悬液，还可以为热水提取菌体混悬液而得的提取液。优选仅包含乳酸菌即可。作为死菌体，可举出通过以下方式而得到的死菌体混悬液、其干燥物等：将通过常规方法进行培养、收集的乳酸菌的菌体进行清洗、离心脱水，根据需要反复进行清洗和脱水后，悬浮于蒸馏水、生理食盐水等，将该混悬液例如以80～115℃加热30分钟～3秒。作为上述死菌体混悬液的干燥方法，可以使用公知的干燥方法，没有特别限定，可以使用喷雾干燥、冷冻干燥等方法。另外，也可以根据需要在基于加热等的杀菌处理前后或者干燥处理前后进行酶处理、表面活性剂处理、磨碎·粉碎处理。
本实施方式的精氨酸酶的产生促进剂由于具有肝脏细胞的精氨酸酶的产生促进能力，因而实现尿素循环的活化，进而实现氨代谢的促进，因此对高氨血症的抑制(症状的缓和)是有用的。另外，由于具有皮肤细胞的精氨酸酶的产生促进能力，因此皮肤组织的尿素含量增加，其结果，实现皮肤的软化，而且皮肤组织的保水性得到改善。另外，由于通过所产生的精氨酸酶而抑制黑色素产生，因此作为美白剂也是有用的。
本实施方式的精氨酸酶的产生促进剂可以作为外用组合物使用，也可以作为口服用组合物使用。具体而言，可以作为药品、准药品、局部或全身用的皮肤化妆品、以用于头皮和头发的药用或化妆用制剂类为代表的各种皮肤外用剂、沐浴剂、口服摄取的药品和准药品的内服用组合物、一般的饮食品、营养补充剂、饮料、功能性食品等食品组合物等各种形式使用。
本实施方式的精氨酸酶的产生促进剂所含有的乳酸菌的数量根据各形式，另外根据其剂型、用途而适当地调整即可，没有特别限制，一般，通常悬浮调整为每克含有1×106个以上，优选为1×107个以上，更优选为1×108个以上，进一步优选为1×109个以上。
作为本实施方式中使用的乳酸菌，优选使用该乳酸菌的混悬液。混悬液中的乳酸菌的数量没有特别限定，通常，为每克1×109个以上即可，优选每克1×1010个以上，更优选每克1×1011个以上，进一步优选每克1×1012个以上。本实施方式的精氨酸酶产生促进剂中配合乳酸菌的混悬液的量通常为0.00001重量％～20重量％，优选0.00005重量％～10重量％，更优选0.0001重量％～5重量％，进一步优选0.0005重量％～2重量％，特别优选0.001重量％～1重量％。
本实施方式的精氨酸酶产生促进剂的发明中除了乳酸菌以外，还可以在不损害本实施方式的效果的范围包含其它成分。例如，可以与通常的对外用或内用原料的处理等中使用的药剂等组合使用，特别优选通过并用药剂而更容易表现出本实施方式的效果的组合。作为其它成分，可举出油性成分、润滑剂、赋形剂、粘合剂、崩解剂等。另外，也可以适当适量地含有甜味剂、酸味剂、香味剂、着色剂、色素等添加物。应予说明，本实施方式的上述各种组合物、各种药剂中，这些成分可以分别单独使用1种，也可以并用2种以上。
作为油性成分，可例示各种脂肪酸酯、烃、高级脂肪酸、高级醇等。作为润滑剂，可以例示硬脂酸镁、硬脂酸、硬脂富马酸钠、蔗糖脂肪酸酯、聚乙二醇(Macrogol)、滑石等。
作为粘合剂，可以例示羟丙基纤维素、羟丙基甲基纤维素、甲基纤维素、明胶、淀粉、糊精、阿拉伯胶、海藻酸钠、黄芪胶、精制明胶、聚乙烯醇、聚乙烯吡咯烷酮、甲基纤维素、聚乙烯醇(部分皂化物)、乙基纤维素、支链淀粉、聚乙二醇(Macrogol)等。
作为崩解剂，可以例示羧甲基纤维素钙(Carmellose Calcium)、羧甲基纤维素钠(Carmellose Sodium)、低取代度羟丙基纤维素、羧甲基纤维素、羧甲基淀粉钠、交联型羧甲基纤维素钠(Crosscarmellose Sodium)、粉末纤维素、纤维素或其衍生物、交联型聚乙烯吡咯烷酮(crospovidone)、淀粉、羧甲基淀粉、羟丙基淀粉、琼脂等。
作为赋形剂，可以例示结晶纤维素、海藻酸钠、黄原胶等多糖类、α化淀粉、羟丙基淀粉、玉米淀粉、马铃薯淀粉等淀粉及其衍生物、蔗糖、葡萄糖、木糖醇、赤藓糖醇、山梨糖醇、乳糖醇、海藻糖、帕拉金糖(PALATINOSE)、PALATINT(氢化帕拉金糖)、甘露醇、麦芽糖醇、乳糖醇、乳糖、果糖、粉末还原麦芽糖浆等糖类和糖醇类、粉末纤维素、部分α化淀粉、乙基纤维素等纤维素及其衍生物、轻质无水硅酸、氧化钛、氢氧化铝凝胶、合成硅酸铝、三硅酸铝、二氧化硅、高岭土、可可脂、柠檬酸或其盐、硬脂酸或其盐、磷酸氢钙、磷酸氢钠等。
作为甜味剂，可例示阿斯巴甜、乙酰磺胺酸钾、糖精钠、三氯蔗糖、甜叶菊、索马甜等。作为酸味剂，可例示柠檬酸、琥珀酸、酒石酸、苹果酸、富马酸等。作为香味剂，可以例示薄荷醇、樟脑、冰片、柠檬烯等单萜类、各种香料等。
本实施方式的精氨酸酶的产生促进剂的各种制剂的剂型没有特别限定，可以根据给药方式而适当地选择。口服给药的情况下，可举出片剂、胶囊剂、颗粒剂、散剂、细粒剂、糖浆剂、缓释片、片剂(tablet)、咀嚼片剂或滴剂等。
本实施方式的精氨酸酶的产生促进剂另外在作为外用剂使用的情况下也可以根据其应用部位而适当地配合油性成分、保湿成分等。
用于皮肤外用剂的情况下，其剂型没有特别限定。作为制剂，例如，可举出乳霜、乳液、油、化妆水、面膜、水性凝胶、油凝胶和软膏等。从经皮吸收性的方面出发，乳霜、乳液、油等可以说是更优选的剂型。这些皮肤外用剂可以根据剂型而适当地选择并配合上述三萜化合物，除此以外，通过与通常的皮肤外用剂同样的方法进行制造。
另外，皮肤外用剂中，除了乳酸菌以外，还可以适当地选择配合通常用于皮肤外用剂的液体石蜡、凡士林、角鲨烷等烃类、肉豆蔻酸异丙酯(IPM)、合成鲸蜡、荷荷巴油、巴西棕榈蜡等酯类、橄榄油、牛脂等动植物油脂、鲸蜡醇、硬脂醇等高级醇类、硬脂酸、油酸等高级脂肪酸类、十二烷基硫酸钠、烷基磺基琥珀酸酯等阴离子表面活性剂、烷基季胺盐等阳离子表面活性剂、脂肪酸单甘油酯、聚氧乙烯氢化蓖麻油等非离子表面活性剂、烷基甜菜碱等两性表面活性剂等表面活性剂类、甘油、丙二醇等多元醇类、乙醇、丙醇等低级醇类、对羟基苯甲酸酯类、葡萄糖酸氯己定等防腐剂类、对氨基苯甲酸衍生物、二苯甲酮衍生物等紫外线吸收剂、维生素E、丁基羟基甲苯等抗氧化剂、阿拉伯胶、羧基乙烯基聚合物等增稠剂、聚乙二醇等保湿剂、柠檬酸盐、乙酸盐等pH调节剂、氧化钛、硅胶、滑石等粉体类、香料、色素等、透明质酸、胎盘提取物、高丽参提取物、维生素类、甾醇糖苷等与各种目的对应的药效成分等。
本实施方式的酶活化剂能够使尿素循环活化，因此也可以作为尿素循环活化剂利用。应予说明，本实施方式中使尿素循环活化是指尿素循环的各个酶的表达增加、生物体中的构成尿素循环的各个酶作用的程度比以往增加、代谢整体得到促进等。
本实施方式的精氨酸酶的产生促进剂通过活化精氨酸酶来促进氨的代谢(解毒)，因此作为用于改善(症状缓和)高氨血症的症状的高氨血症改善剂也是有用的。
将本实施方式的精氨酸酶的产生促进剂作为口服剂进行给药时，可以根据乳酸菌的配合浓度、剂型、给药对象者的年龄、体重、性别、在运动前服用的情况下根据运动的负荷等条件而适当地决定。例如剂型为片剂的情况下，优选与水等一同服用。给药间隔可以适当地决定，例如，可以为饭前、饭后、饭间、运动前、运动后中的任一者。
本实施方式的精氨酸酶的产生促进剂的口服给药量以每1天的人的乳酸菌摄取量计，通常为1mg～5000mg/天，优选为50mg～1000mg/天，进一步优选为100mg～500mg/天。
将本实施方式的精氨酸酶的产生促进剂作为外用剂进行给药时，可以根据乳酸菌的配合浓度、剂型、给药对象者的年龄、体重、性别、应用部位等条件而适当地决定。例如外用剂为乳霜的情况下，1天1～3次涂布于皮肤。
将本实施方式的精氨酸酶的产生促进剂作为外用剂进行给药时，在外用剂中以成为0.01～100μg/ml浓度的方式进行配合。优选为0.05～50μg/ml，特别优选为0.1～10μg/ml。
实施例
以下，对具体的实施例进行说明，但本发明并不限定于下述的实施例。
实施例1(乳酸菌混悬液的制备)
将乳酸乳球菌(Lactococcus lactis)LLA-34菌株用由市售的MRS粉末培养基(Difuco&BBD公司制)制备的MRS液体培养基在37℃下静置培养24小时。将得到的预培养液接种于新的MRS液体培养基，在37℃下静置培养24小时。培养结束后，通过7000rpm的离心分离来分离并回收菌体。将该操作重复多次，将菌体用蒸馏水清洗。接着，将清洗后的菌体以达到光学浓度(OD)＝40的方式再悬浮于蒸馏水，以121℃进行20分钟杀菌处理，得到乳酸菌混悬液。
＜试验1：乳酸菌的精氨酸酶产生促进能力评价试验(人角质细胞)＞
1.试验方法
将人角质细胞(PHK16-0b)以2.0×105个细胞/孔接种于6孔板，用在EpiLife(Gibco公司制)中添加了Human KG增殖添加剂(Gibco公司制)和100ug/ml青霉素-链霉素的培养基培养3天。作为检体(实施例1)的乳酸菌混悬液以在培养基中为OD＝0.08(浓度1)、OD＝0.4(浓度2)的方式添加，培养4天。将细胞用PBS(-)清洗后，用100μl的Passive LysisBuffer(Gibco公司制)将细胞裂解。将本细胞裂解液离心，使用QuantiChrom精氨酸酶测试盒(QuantiChrom Arginase Assay Kit，Bio assay Systems公司制)按照随附的操作方法来测定所得到的上清液的精氨酸酶活性。利用读板器(Perkin Elmer公司制，ARVO X3)由430nm的吸收而算出尿素量。另外，用蛋白质检测试剂盒(Takara Bio公司制)来测定细胞裂解液中的总蛋白质量，将每单位细胞蛋白质的尿素量换算为细胞内精氨酸酶活性。将未添加试验检体(比较例1：对照)计为100％而算出试验检体添加时的细胞内精氨酸酶活性。
2.结果
将测定结果示于下述表1。
[表1]
根据表1可知乳酸乳球菌(Lactococcus lactis)LLA-34菌株使人角质细胞的精氨酸酶活性增加。角质细胞存在于人的皮肤中，可以确认皮肤中的精氨酸酶活性提高。这表示皮肤的保湿、代谢改善。
＜试验2：乳酸菌的精氨酸酶产生促进能力评价试验(人肝癌细胞)＞
1.试验方法
将人肝癌细胞(HepG2)以2.0×105个细胞/孔接种于6孔板，用添加了10％的胎牛血清、100μg/ml的青霉素-链霉素的HepG2专用培养基(CET公司制)培养1天。作为检体(实施例1)的乳酸菌混悬液以在培养基中为OD＝0.08(浓度1)、OD＝0.4(浓度2)的方式添加，培养1天。细胞裂解液的制备、细胞内精氨酸酶活性量的测定按照与试验1同样的方法进行。将未添加试验检体(比较例1)计为100％而算出试验检体添加时的细胞内精氨酸酶活性。
2.结果
将测定结果示于下述表2。
[表2]
根据表2可知乳酸乳球菌(Lactococcus lactis)LLA-34菌株使人肝癌细胞的精氨酸酶活性增加。这些菌株不仅对皮肤中的精氨酸酶活化有效，也对肝脏中的精氨酸酶活化也有效。
<试验3：精氨酸酶产生促进能力评价试验＞
为了验证实施例1的效果而实施比较例2的精氨酸酶产生促进能力评价试验。
比较例2：袈裟柿皮(冷冻干燥粉体)，将市售的袈裟柿的皮冷冻干燥后进行粉碎而得的粉体
1.试验方法
将人角质细胞(PHK16-0b)以2.0×105个细胞/孔接种于6孔板，用在EpiLife(Gibco公司制)中添加了Human KG增殖添加剂(Gibco公司制)和100μg/ml青霉素-链霉素的培养基培养3天。作为检体的比较例2以在培养基中终浓度为100ug/m1的方式添加，培养4天。细胞裂解液的制备、细胞内精氨酸酶活性量的测定利用与试验1同样的方法进行。将未添加试验检体(比较例1)计为100％而算出试验样品添加时的细胞内精氨酸酶活性。
2.结果
将测定结果与试验1(实施例1)的结果一并示于下述表3。
[表3]
| 细胞内精氨酸酶活性量(％) | |
| 比较例1(无添加) | 100 |
| 比较例2 | 78 |
| 实施例1 | 234 |
虽然可知比较例2具有肤质、肝脏的功能改善效果，但表明实施例1的乳酸菌在精氨酸酶产生促进能力上具有更高的效果。
产业上的可利用性
本申请作为精氨酸酶产生促进剂而具有产业上的可利用性。
Claims (8)
1.一种精氨酸酶的产生促进剂，以乳酸菌为有效成分。
2.根据权利要求1所述的产生促进剂，其中，促进皮肤组织的所述精氨酸酶的产生。
3.根据权利要求1或2所述的产生促进剂，其中，所述乳酸菌包含乳球菌属。
4.根据权利要求1～3中任一项所述的产生促进剂，其中，将为死菌体的所述乳酸菌作为有效成分。
5.一种皮肤软化剂，以乳酸菌为有效成分。
6.一种肝脏组织的精氨酸酶的产生促进剂，以乳酸菌为有效成分。
7.一种尿素循环活化剂，以乳酸菌为有效成分。
8.一种高氨血症改善剂，以乳酸菌为有效成分。
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