CN106999521A - 老化抑制剂 - Google Patents
老化抑制剂 Download PDFInfo
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- CN106999521A CN106999521A CN201580067409.8A CN201580067409A CN106999521A CN 106999521 A CN106999521 A CN 106999521A CN 201580067409 A CN201580067409 A CN 201580067409A CN 106999521 A CN106999521 A CN 106999521A
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Abstract
本发明的课题在于,提供:延迟老化症状的发现、且带来寿命的延长、安全性高的老化抑制剂。解决上述课题的本发明的老化抑制剂的特征在于,含有植物发酵物作为有效成分,所述植物发酵物为如下(a)~(g)的混合物,(a)选自由大麦、黑豆、红米、黑米、小豆、薏仁、稗子、小米和黄米组成的组中的1种或2种以上的豆/谷物类的曲霉发酵物;(b)选自由蜜柑、葡萄、苹果、山葡萄、桃子、柿子、木瓜、梨、西瓜、梅子、无花果、榅桲、南瓜、金橘、柚子、枇杷、杏、枣、栗子、葛枣和李子组成的组中的1种或2种以上的水果类的酵母和/或乳酸菌发酵物;(c)选自由紫薯、洋姜、胡萝卜、洋葱、甘薯、芋头、薯蓣、萝卜、红芜菁、牛蒡、莲藕、雪莲果、百合根、慈菇、姜、大蒜和姜黄组成的组中的1种或2种以上的块根类/薯类的酵母和/或乳酸菌发酵物;(d)选自由卷心菜、紫苏、桑叶、鱼腥草、苦艾、山白竹和蒲公英组成的组中的1种或2种以上的花/叶菜类的酵母和/或乳酸菌发酵物;(e)选自由海带、裙带菜和海蕴组成的组中的1种或2种以上的海藻类的酵母和/或乳酸菌发酵物;(f)选自由黑芝麻、胡桃和银杏组成的组中的1种或2种以上的种子类的酵母和/或乳酸菌发酵物;(g)选自由灰树花和香菇组成的组中的1种或2种的蘑菇类的酵母和/或乳酸菌发酵物。
Description
技术领域
本发明涉及利用了植物发酵物的老化抑制剂,详细而言,涉及兼具伴随着动物的老龄的老化症状的体现抑制作用与存活的延长作用、且安全性优异的老化抑制剂。
背景技术
老化对动物而言是不可避免的现象。随着生命科学的进步,对于老化的研究推进,已经阐明了个体、器官、组织、细胞、基因水平、进而与其连带的神经·血管、免疫调节、激素类、调节因子的各种物质组的协调作用,但是详细的机制仍未阐明。然而,发现了其线索,线虫、果蝇、大鼠、小鼠、猴子等动物中,广泛已知的是,饮食热量限制显示出寿命延长效果(非专利文献1~4)。作为其背景,推定线粒体中的能量代谢参与。另外为了老化抑制·寿命延长,也探索了热量限制模拟物(非专利文献5)。另外胰岛素·生长因子(IGF-1)信号与寿命的相关以线虫、果蝇、哺乳类(小鼠)的共通信号传导通路形式体现(非专利文献6)。进而,还发现了作为红葡萄的低分子成分的白藜芦醇期待老化抑制作用、寿命延长效果的结果(非专利文献7),进行了类似物质的探索研究(非专利文献8)。最近,去乙酰化酶具有组蛋白的脱乙酰化酶活性,发现了参与寿命延长,期待将各种线索的相关。另外还发现了去乙酰化酶的低分子调节物质(非专利文献9)。
可能难以阻止伴随着老化而引起的各种老化症状,但可以期待延迟老化症状的体现、进而延长寿命。然而,预测这些老化症状的原因与多个基因、过程相关(非专利文献10)。因此,老化症状的发生延迟、寿命的延长的评价必需基于动物个体的试验。另外,为了寿命的试验研究,需要世代时间短的模型动物。作为这样的模型动物,有线虫、果蝇、小鼠,其中,小鼠为哺乳动物,正常型小鼠也为2~3年左右的寿命,因此,在较短期间内,可以推定对老化·寿命的效果。京都大学的动物实验设施中,AKR系的小鼠中发现的老化促进模型小鼠即SAM-P系小鼠的寿命为1.5年左右,且显示出各种老化特有的症状,因此,与同系的正常型的SAM-R1小鼠相比,大多用于老化研究。这些小鼠也已经由SAM研究会进行了传代繁殖·市售,老化症状的评价方法也由SAM研究会进行了标准化。标准化的老化指标利用行动异常、缓慢化、眼症状(眼睛周围的损害、角膜的白浊、白内障等)、脱发、毛长等皮肤的老化症状、背骨的弯曲。另一方面,作为老化抑制的一个指标的存活率的延长未包含于SAM研究会的标准的评价指标。
由辐射线照射所导致的障碍中,观察到与老化类似的症状(非专利文献11)。6~14戈瑞(gray)的X射线照射过的小鼠的小肠的粘膜干细胞受到障碍,但伊藤等人报道了,以混合饲料给予了味噌的小鼠中,小肠隐窝的干细胞的损伤少(非专利文献12)。然而,没有关于老化症状、存活率的报道。虽然报道了使用同样的评价系,作为植物发酵食品的万田酶对X射线照射过的小鼠的小肠的粘膜干细胞障碍抑制是有效的(非专利文献13),但是没有报道老化症状的抑制、存活率的延长。
进而,报道了使用SAM-P1系的老化促进模型小鼠的辅酶Q10和还原型的老化症状的抑制效果(非专利文献14)。报道了,与CoQ10无添加组相比,在眼症状、皮肤症状、背骨弯曲和综合症状分数上显示出显著的老化抑制效果,但是,未见寿命的延长。另外将大豆蛋白SPI与酪蛋白比较,老化度未见差异,但是SPI确认到存活率延长效果(非专利文献15)。
关于参与老化的去乙酰化酶,报道了,由中药等引起去乙酰化酶的增强、抑制,但没有进行寿命延长的试验。另一方面,报道了,红葡萄中所含的白藜芦醇延长果蝇、小鼠的寿命。
此外,关于老化抑制或者寿命延长,进行了各种报道,例如公开了,通过应用妨碍编码G蛋白质γ亚基11的基因的表达或其作用的物质,可以抑制细胞老化(专利文献1)。另外报道了,具有特定结构的烷基间苯二酚类具有去乙酰化酶活性化作用和老化抑制作用,显示出延长个体寿命的效果(专利文献2)。进而公开了,蜂王浆、特定结构的杂环化合物、白藜芦醇、葡萄叶提取物、活性炭、紫荆提取物、姜黄素类、葛根提取物等显示出防止老化、寿命延长等作用(专利文献3~10)。
然后,实际情况是,对于哺乳动物,基本没有均发挥老化症状的体现延迟效果和位于老化的延长线上的寿命的延长效果的食品、药品等的报道。
现有技术文献
专利文献
专利文献1:国际公开2007/102572
专利文献2:日本特开2013-249260号公报
专利文献3:日本特开2012-207004号公报
专利文献4:日本特开2008-94795号公报
专利文献5:日本特开2007-145809号公报
专利文献6:日本特开2010-208969号公报
专利文献7:日本特表2006-515590号公报
专利文献8:日本特开2008-120726号公报
专利文献9:日本特开2010-270012号公报
专利文献10:日本特开2012-246242号公报
非专利文献
非专利文献1:A.Dillin et al,“Rate of behavior and aging specified bymitochondrial function during development”,Science 298,2398-2401(2002)
非专利文献2:Grandison RC1,Piper MD,Partridge L.“Amino-acid inbalanceexplains extension of life span by dietary restriction in Drosophila”,Nature462,1061-1064(2009)
非专利文献3:“认识老化研究”羊土社2002、井出利宪编、P25,37
非专利文献4:Coleman R.J.et al,“Carolic restriction delays diseaseonset and mortality in Rhesus monkeys”,Science 325,201-204(2009)
非专利文献5:“热量限制模拟物”千叶卓哉、下川功、实验医学增刊31-No.20,182-189(2013)
非专利文献6:“胰岛素·IGF-1信号与老化·寿命控制”植木浩二郎、实验医学增刊31-No.20,22-27(2013)
非专利文献7:“Resveratorol improves health and survcival of mice on ahigh-calorie diet”Bauer et al:Nature,477:337-342(2006)
非专利文献8:“源自红葡萄酒的多酚Protpcathchuic acid对果蝇寿命的效果”八木勇三等、Food Function、11、9-13(2013)
非专利文献9:“去乙酰化酶的低分子调节物质”D.A.Sinclair、B.P.Hubbard,实验医学增刊31-No.20,209-217(2013)
非专利文献10:“老化·寿命的科学”实验医学增刊31卷-No.20(2013)今井真一郎、吉野纯编集、羊土社
非专利文献11:Hoeijmakers,J.H.J.:“Genome maintenance mechanisms forpreventing cancer”:Nature,411:366-374,2001
非专利文献12:伊藤明弘“味噌去除放射性物质的效用预防发癌的味噌的生理作用”“味噌科学最前线”1999味噌健康推广委员会发行
非专利文献13:“植物发酵物对X线照射的防御效果-对小肠的隐窝再生的影响-”芦田等、日本未病系统学会杂志、12(1)129-130(2006)
非专利文献14:“Reduced coenzyme Q10supplementation deceleratesenescence in SAM-P1mice”J.Yan et.al.:Experimental Gerontology 41(2006)130-140
非专利文献15:“针对基于大豆蛋白的促进老化抑制效果-基于老化促进模型小鼠的实验的研究”河野笃子等、Nutr.Sci.Soy Protein、Jpn:7,25-29(1986)
非专利文献16:“老化解析”簗濑澄乃、石井直明、“线虫实验室手册”三谷昌平编、p.201-206(2003)Springer-Verlag Tokyo,Inc.
发明内容
发明要解决的问题
本发明鉴于上述实际情况,其课题在于,提供:使老化相关的多个症状的体现延迟、且带来寿命的延长、安全性高的老化抑制剂。
用于解决问题的方案
本发明人为了解决上述课题,进行了深入鋭意研究,结果发现:使有多种食用经验的植物用乳酸菌、酵母、曲霉等发酵而成的发酵物抑制伴随着老龄的各种老化症状,且显示出存活率的延长效果,至此完成了本发明。
即,本发明为一种老化抑制剂,其特征在于,含有植物发酵物作为有效成分,所述植物发酵物为如下(a)~(g)的混合物,
(a)选自由大麦、黑豆、红米、黑米、小豆、薏仁、稗子、小米和黄米组成的组中的1种或2种以上的豆/谷物类的曲霉发酵物;
(b)选自由蜜柑、葡萄、苹果、山葡萄、桃子、柿子、木瓜、梨、西瓜、梅子、无花果、榅桲、南瓜、金橘、柚子、枇杷、杏、枣、栗子、葛枣和李子组成的组中的1种或2种以上的水果类的酵母和/或乳酸菌发酵物;
(c)选自由紫薯、洋姜、胡萝卜、洋葱、甘薯、芋头、薯蓣、萝卜、红芜菁、牛蒡、莲藕、雪莲果、百合根、慈菇、姜、大蒜和姜黄组成的组中的1种或2种以上的块根类/薯类的酵母和/或乳酸菌发酵物;
(d)选自由卷心菜、紫苏、桑叶、鱼腥草、苦艾、山白竹和蒲公英组成的组中的1种或2种以上的花/叶菜类的酵母和/或乳酸菌发酵物;
(e)选自由海带、裙带菜和海蕴组成的组中的1种或2种以上的海藻类的酵母和/或乳酸菌发酵物;
(f)选自由黑芝麻、胡桃和银杏组成的组中的1种或2种以上的种子类的酵母和/或乳酸菌发酵物;
(g)选自由灰树花和香菇组成的组中的1种或2种的蘑菇类的酵母和/或乳酸菌发酵物。
另外本发明为一种饮食品,其含有上述老化抑制剂。
另外本发明为一种寿命延长剂,其含有上述(a)~(g)的植物发酵物作为有效成分。
发明的效果
本发明的老化抑制剂可以抑制伴随着动物的老龄的宽范围的老化症状的体现,并且带来存活率延长。另外,作为有效成分即植物发酵物的原料的植物和微生物的食用经验均丰富,因此,安全性极高。
附图说明
图1为制造例2的植物发酵物中所含的多酚类的基于HPLC的分析结果。
图2为制造例2的植物发酵物中所含的白藜芦醇的基于HPLC的分析结果。
图3为示出试验例1中的氧敏感性线虫变异株(mev-1)在氧气100%气氛下的存活曲线的图。纵轴表示每3.5cm有盖培养皿的线虫的平均存活只数,横轴表示培养天数。
图4为示出试验例2中的SAM小鼠的存活曲线的图。纵轴表示SAM小鼠的存活率(%),横轴表示SAM小鼠的周龄。
图5为示出试验例2中、对于植物发酵物2%和0.2%给予试验的存活率、基于Kaplan-Meier解析的显著性检验的结果的图。纵轴表示累积存活率,横轴表示周龄。实线为植物发酵物2%组的存活曲线,虚线为植物发酵物0.2%组的存活曲线。
图6为示出试验例2中、使用SAM研究会的标准法的老化度的综合评价分数的推移的图。纵轴表示老化指标综合分/只,横轴为周龄。实线为植物发酵物2%组,虚线为植物发酵物0.2%组。为了进行显著性检验,选择能够确保存活数为6/8只以上的54周龄和60周龄的2点,用箭头表示。
图7为试验例2中、针对54周龄(A)和60周龄(B)、分别比较植物发酵物2%组与0.2%组的老化度综合评价分数的图。
图8为试验例2中、针对54周龄(A)和60周龄(B)、比较植物发酵物2%组与0.2%组的植物发酵物的老化指标之一即“背骨弯曲分数”的图。
图9为示出试验例2中、针对正常型(SAM-R1)和老化促进型(SAM-P1)小鼠、主要在鼠颈部产生的肿瘤的体积的推移的图。
具体实施方式
本发明的老化抑制剂含有下述植物发酵物作为有效成分。该植物发酵物为以下(a)~(g)的植物发酵物的混合物。
(a)通过使曲霉作用于选自由大麦、黑豆、红米、黑米、小豆、薏仁、稗子、小米和黄米组成的组中的1种或2种以上的豆/谷物类而得到的发酵物。
(b)通过使酵母和/或乳酸菌作用于选自由蜜柑、葡萄、苹果、山葡萄、桃子、柿子、木瓜、梨、西瓜、梅子、无花果、榅桲、南瓜、金橘、柚子、枇杷、杏、枣、栗子、葛枣和李子组成的组中的1种或2种以上的水果类而得到的发酵物。
(c)通过使酵母和/或乳酸菌作用于选自由紫薯、洋姜、胡萝卜、洋葱、甘薯、芋头、薯蓣、萝卜、红芜菁、牛蒡、莲藕、雪莲果、百合根、慈菇、姜、大蒜和姜黄组成的组中的1种或2种以上的块根类/薯类而得到的发酵物。
(d)通过使酵母和/或乳酸菌作用于选自由卷心菜、紫苏、桑叶、鱼腥草、苦艾、山白竹和蒲公英组成的组中的1种或2种以上的花/叶菜类而得到的发酵物。
(e)通过使酵母和/或乳酸菌作用于选自由海带、裙带菜和海蕴组成的组中的1种或2种以上的海藻类而得到的发酵物。
(f)通过使酵母和/或乳酸菌作用于选自由黑芝麻、胡桃和银杏组成的组中的1种或2种以上的种子类而得到的发酵物。
(g)通过使酵母和/或乳酸菌作用于选自由灰树花和香菇组成的组中的1种或2种的蘑菇类而得到的发酵物。
本发明的老化抑制剂可以直接将混合有上述(a)~(g)的发酵物的植物发酵物作为有效成分,通过使其进一步多阶段发酵,从而可以提高呈味性、制剂性。
作为酵母,可以举出:属于酵母属、接合酵母属等的酵母,其中,优选使用酿酒酵母(S.cervisiae)、鲁氏酵母(Z.rouxii)、啤酒酵母(S.exiguus)等。作为乳酸菌,例如可以举出:属于片球菌(Pediococcus)属、明串珠菌(Leuconostoc)属、乳酸菌(Lactobacillus)属、乳球菌(Lactococcus)属等的乳酸菌,其中,优选使用乳酸片球菌(P.acidilacti)、短乳杆菌(L.brevis)、肠系膜明串珠菌(L.mesenteroides)、植物乳杆菌(L.plantarum)、乳酸乳球菌(L.lactis)、清酒乳杆菌(L.sakei)、戊糖片球菌(P.pentosaceus)、干酪乳杆菌(L.casei)、罗伊氏乳杆菌(L.reuteri)和弯曲乳杆菌(L.curvatus)等,可以使用它们中的1种或2种以上。作为曲霉,可以举出:米曲霉(Aspergillus oryzae)、黑曲霉(Aspergillusniger)、白曲霉(Aspergillus kawachii)等,可以使用它们中的1种或2种以上。它们也可以使用市售品。
供至发酵的豆/谷物类、水果等植物可以直接使用,或者也可以根据需要进行粉碎、干燥等前处理。另外也可以根据需要添加水进行稀释。
上述(a)~(g)的发酵物通过向作为原料的植物中接种乳酸菌、酵母或曲霉并使其培养而得到。培养只要通过常规方法进行即可,例如,可以对于1种或2种以上的植物的混合物,添加0.001~1质量%左右的乳酸菌、酵母或曲霉,在20~50℃下进行70~140小时左右的发酵处理。
也可以将如此得到的(a)~(g)的发酵物混合而直接作为有效成分,优选的是,根据需要进而在20~40℃下进行200~300小时左右的培养。进一步优选使该混合物后发酵(熟化)。后发酵可以根据需要使醋酸菌作用,例如,可以向使上述酵母作用于上述(a)~(g)所含的植物中的1种或2种以上而得到的物质中,添加使醋酸杆菌(A.aceti)等醋酸菌作用的醋酸发酵物。后发酵可以在25~35℃下进行70小时~约1年左右。通过进行基于后发酵的熟化过程,可以改善呈味性、制剂性,另外也可以实现抗氧化活性的增大。
作为用于制造上述植物发酵物的适合的方法的例子,可以举出:多阶段复合发酵方式。该方法如下进行:将以水果类、蔬菜类、海藻类为主原料的复合乳酸菌发酵物与以蔬菜类、块根类、种子类、蘑菇类、水果类为主要原料的酵母发酵物混合,向其中加入以谷物类、豆类为主原料的曲霉发酵物,进而加入醋酸发酵物并混合,然后进行过滤浓缩,进而进行约1年左右的后发酵。可以认为,通过这样的多阶段复合发酵,进而利用其他类型的菌类使各发酵产物其同化·转化,可以提高风味且提高抗氧化活性等效果。
本发明的老化抑制剂和寿命延长剂可以如下得到:依据公知的制剂学制法,向如此得到的植物发酵物中加入药理学上能够允许的载体、赋形剂、水分活性调节剂等并制剂化而得到。植物发酵物可以根据需要浓缩而调整浓度,或者通过喷雾或冷冻干燥等进行粉末化。作为制剂化中使用的载体、赋形剂,例如可以使用乳糖、葡萄糖、蔗糖、淀粉、糖类混合物等。作为最终形态,可以使用溶液、糊剂、软胶囊、咀嚼片、胶囊。作为用量·用法,例如,以有效成分的植物发酵物计,大人每1天可以口服摄取0.1~10g、优选口服摄取0.6~6g(换算为固体成分)左右。
另外,本发明的老化抑制·寿命延长剂除药品、准药品等之外,也可以与公知的食品原材料一起配合而形成饮食品的形态。上述植物发酵物也可以直接摄取,为了提高保存性,也可以在杀菌后进行过滤、浓缩,或者根据需要添加赋形剂进行喷雾或冷冻干燥而形成粉末状。进而,为了提高流通过程中的保存期限,理想的是,进行浓缩而降低水分活性。作为上述饮食品的形态,可以举出:糊剂、软胶囊、片剂、保健饮料等。大人每1天,以植物发酵物计,摄取0.1~10g、优选0.6~6g(换算为固体成分)左右,从而可以得到优异的老化抑制效果等。作为使这样的上述植物发酵物制剂化、食品的形态的市售品,可以举出:天生酶金印、天生酶胶囊(Nihon Sizen Hakkoh制)等。
本发明中使用的有效成分的植物发酵物具有下述(1)至(4)的性质。
(1)呈味性
具有源自水果、蔬菜等原料的甜味、在有机酸基础上的源自谷芽的甜味。虽然包含源自原料的多酚类,但是苦味少。
(2)对水的溶解性
容易溶解于水。
(3)稳定性
对热、酸稳定,糊剂在室温下保存1年,也不会腐败、呈味性也没有变化。
(4)安全性
原料中使用的蔬菜、水果、药草等是日常食用的,另外发酵中使用的酵母、乳酸菌、曲霉均使用源自食品的酿造、发酵等的菌种,因此,食用经验丰富。
另外,本发明中使用的植物发酵物具有下述(i)至(vi)的性质。
(i)一般成分(每100g)
(ii)氨基酸组成(每100g)
(iii)有机酸组成(每100g)
(iv)矿物组成(每100g)
(v)多酚(μg/g)
(vi)白藜芦醇
白藜芦醇的反式体与顺式体的含量的总计为0.1mg/g以下。
以该植物发酵物为有效成分的老化抑制剂显示出如下效果:用量依赖性地提高氧敏感性的线虫和老化促进小鼠的存活率,并且使小鼠的伴随着老龄的行动、皮肤、眼、背骨中的老化症状的体现延迟。
实施例
接着,列举实施例等,对本发明进行更详细地说明,但本发明不受这些实施例等的任何限制。
制造例1
植物发酵物的制造(1):
使用以下的植物作为原料。
(a)豆/谷物类(大麦、黑豆、红米、黑米、小豆、薏仁、稗子、小米、黄米)
(b)水果类(蜜柑、葡萄、苹果、山葡萄、桃子、柿子、木瓜、梨、西瓜、梅子、无花果、榅桲、南瓜、金橘、柚子、枇杷、杏、枣、栗子、葛枣、李子)
(c)块根类(紫薯、洋姜、胡萝卜、洋葱、甘薯、芋头、薯蓣、萝卜、红芜菁、牛蒡、莲藕、雪莲果、百合根、慈菇、姜、大蒜、姜黄)
(d)花/叶菜类(卷心菜、紫苏、桑叶、鱼腥草、苦艾、山白竹、蒲公英)
(e)海草类(海带、裙带菜、海蕴)
(f)种子类(黑芝麻、胡桃、银杏)
(g)蘑菇类(灰树花、香菇)
将以乳酸菌类(乳酸片球菌(P.acidilacti)、短乳杆菌(L.brevis)、肠系膜明串珠菌(L.mesenteroides)、植物乳杆菌(L.plantarum)、乳酸乳球菌(L.lactis)、清酒乳杆菌(L.sakei)、干酪乳杆菌(L.casei))成为约1.0×105cfu/g的菌浓度的方式调制的培养液0.2质量%接种至上述(c)、(d)和(g)的原料(730g),在30℃下进行50小时培养。另外将以酵母类(酿酒酵母(S.cervisiae)5种、鲁氏酵母(Z.rouxii)2种)成为约1.0×105cfu/g的菌浓度的方式调制的培养液0.4质量%接种至上述(b)、(e)和(f)的原料(900kg),在30℃下进行50小时培养。将曲霉类(黄曲霉、黑曲霉、白曲霉)0.1质量%接种至(a)豆类和谷物类系原料(1000kg),在35℃下进行70小时培养。接着,将各培养物混合后,在30℃下进行200小时培养。对结束了发酵的醪液进行固液分离操作,将所得滤液浓缩而形成糊状的物质分别注入至容器,1年后进一步使其发酵(熟化),得到植物发酵物。
制造例1中得到的植物发酵物具有以下的性质。
(i)一般分析值(每100g)
(ii)氨基酸组成(每100g)
通过氨基酸自动分析计对用6N盐酸水解试样而得到的物质进行分析。对于胱氨酸,过甲酸氧化处理后,使用盐酸水解。色氨酸使用高效液相色谱法。
(iii)有机酸组成(每100g)
(iv)矿物组成(每100g)
制造例2
植物发酵物的制造(2):
使用以下的植物作为原料。
(a)豆/谷物类(大麦、黑豆、红米、黑米、小豆、薏仁、稗子、小米、黄米)
(b)水果类(蜜柑、葡萄、苹果、山葡萄、桃子、柿子、木瓜、梨、西瓜、梅子、无花果、榅桲、南瓜、金橘、柚子、枇杷、杏、枣、栗子、葛枣、李子)
(c)块根类(紫薯、洋姜、胡萝卜、洋葱、甘薯、芋头、薯蓣、萝卜、红芜菁、牛蒡、莲藕、雪莲果、百合根、慈菇、姜、大蒜、姜黄)
(d)花/叶菜类(卷心菜、紫苏、桑叶、鱼腥草、苦艾、山白竹、蒲公英)
(e)海草类(海带、裙带菜、海蕴)
(f)种子类(黑芝麻、胡桃、银杏)
(g)蘑菇类(灰树花、香菇)
将以乳酸菌类(乳酸片球菌(P.acidilacti)、短乳杆菌(L.brevis)、肠系膜明串珠菌(L.mesenteroides)、植物乳杆菌(L.plantarum)、乳酸乳球菌(L.lactis)、清酒乳杆菌(L.sakei)、干酪乳杆菌(L.casei))成为约1.0×105cfu/g的菌浓度的方式调制的培养液0.2质量%接种至上述(c)、(d)和(g)的原料(730g),在30℃下进行50小时培养。另外将以酵母类(酿酒酵母(S.cervisiae)5种、鲁氏酵母(Z.rouxii)2种)成为约1.0×105cfu/g的菌浓度的方式调制的培养液0.4质量%接种至上述(b)、(e)和(f)的原料(900kg),在30℃下进行50小时培养。将曲霉类(黄曲霉、黑曲霉、白曲霉)0.1质量%接种至(a)豆类和谷物类系原料(500kg),在35℃下进行70小时培养。接着,将各培养物混合后,在30℃下进行200小时培养。对于结束了发酵的醪液进行固液分离操作,将所得滤液浓缩而形成糊状的物质分别注入至容器,1年后进一步使其发酵(熟化),得到植物发酵物。
制造例2中得到的植物发酵物具有以下的性质。
(i)一般分析值(每100g)
(ii)氨基酸组成(每100g)
通过氨基酸自动分析计对用6N盐酸水解试样而得到的物质进行分析。对于胱氨酸,过甲酸氧化处理后,使用盐酸水解。色氨酸使用高效液相色谱法。
(iii)有机酸组成(每100g)
(iv)多酚(参照图1;μg/g)
(HPLC条件)
柱:MCM(4.6x250mm;5μm)
柱温:35℃
流动相:流动相A:100mM磷酸钠、5%甲醇
流动相B:100mM磷酸钠、60%乙腈、10%甲醇
梯度条件:流动相B的直线梯度0-45分钟、0~6%、
流动相B的直线梯度6%~14%、45~60分钟、
流动相B的直线梯度14%~30%、60~90分钟
流动相B的直线梯度30%~40%、90~120分钟、
流动相B的直线梯度40%~77%、120~140分钟、
流动相B的等度洗脱至85%,100分钟
流量:1.0ml/分钟
检测器:CoulArray,Model 5600A
(测定方法)
精确地量取检体约80mg,添加内标液(17-α-雌二醇100μg/mL)100μL,加入90%甲醇溶液5mL,振荡10分钟后,在35℃下进行30分钟超声波提取。提取后,利用离心器(3000rpm,10分钟)进行离心分离后,分取上清,在氮气气流下进行浓缩,然后用0.1M醋酸钠缓冲液定容至2mL。向该溶液1mL中添加β-糖苷酶系酶10mg,在43℃下进行2小时酶处理。向酶处理后的溶液中添加甲醇500μL,用0.45μm过滤器过滤,将所得液体作为试验溶液。
(v)白藜芦醇(参照图2)
含有反式体与顺式体的总计为0.022mg/g的白藜芦醇(反式体0.002mg/g、顺式体0.02mg/g)。
检测器:UV(310nm)
柱:野村化学ODS-MG-3 3×100mm
柱温:40℃
流动相:稀释了的磷酸(1→500)/乙腈混液(4:1)
流量:0.4mL/分钟
(测定方法)
·NSK白藜芦醇(标准溶液)
将1个胶囊(330mg相当于白藜芦醇3.3mg)用70%甲醇溶解,制成100mL。
该液体用0.45μm的膜滤器过滤,将滤液作为标准溶液。
·试样
将试样约1g用水30mL溶解,加入乙酸乙酯20mL,混合,分取上清液,进行减压浓缩。向残留物中加入70%甲醇5mL。将该液体用0.45μm的膜滤器过滤,将滤液作为试样溶液。
红葡萄酒所含的白藜芦醇已知有抗炎症作用、老化抑制作用、或者延长果蝇的寿命。植物发酵物也显示出SAM小鼠中的老化抑制作用和寿命延长作用,因此,为了确认白藜芦醇影响的有无,用HPLC标准品分析其含量。结果,反式体为0.002mg/g、顺式体为0.02mg/g(反式体与顺式体的总量为0.022mg/g)。在该浓度下,无法期待老化抑制效果、寿命延长效果,可以认为,本植物发酵物的老化抑制作用与存活率的延长作用源自其他物质。
试验例1对线虫的延命效果:
将制造例1中得到的植物发酵物用离子交换水稀释,制成2%(w/w)溶液,利用高压釜,以121度进行20分钟灭菌。作为线虫(Caenorhabditis elegans),使用氧敏感性变异株(mev-1)。mev-1株在培养中的氧浓度增加时容易死亡,引起依赖于氧浓度的存活曲线的偏移。证明了,该变异株是源自线粒体的呼吸链中的复合体II的细胞色素b560变异的琥珀酸-辅酶Q脱氢酶的变异,长期间培养而老化了的线虫在体内观察到空洞,进而证明了,伴随着老化的脂褐质蓄积。如此,mev-1变异株是适于老化研究的模型实验动物。存活曲线依据石井等的方法(非专利文献16)来进行。即,将大肠杆菌K562株的用胰蛋白胨肉汤振荡培养的培养液用巴斯德吸管添加1滴至3.5cm Falcon plates中加入的胰蛋白胨琼脂培养基(TN培养基),在30℃下培养1晩时,形成直径5mm左右的大肠杆菌的簇。另行以直径9cm的有盖培养皿的TN琼脂培养基中生长的大肠杆菌为饵,使mev-1生长后,促进生长阶段L4,将总计10只的线虫移植至3.5cm板上的大肠杆菌簇。将移植有线虫的4张3.5cm板放入密封袋后,用氧气进行100%置换。从第二天起每天用实体显微镜记录存活的线虫的数量。观察后,再次用氧气同样地进行置换。如图3所示那样,所得存活曲线在簗濑·石井报道的最大80%氧气饱和的条件下进一步向短寿命侧偏移,表示实验正常进行。氧气100%气氛下的该氧敏感性变异株的寿命与文献(非专利文献16)中报道的簗濑·石井的图相比时,示出50%存活率的培养天数(日龄)比石井等的80%氧气气氛下(约10天)进一步缩短,为约3天。与对照的水组相比,2%植物发酵物组在存活率约50%附近可见延命效果。进行t-检验,结果P=0.025、有显著性差异。可知,mev-1变异株在线粒体的呼吸链中有缺陷,因此强烈地暗示了,活性氧与毒性相关。
试验例2
对于植物发酵物使用老化促进模型小鼠(SAM小鼠)的伴随着老龄的老化抑制效果和存活率的延长效果、和肿瘤的发生抑制效果,如下实施试验。SAM小鼠(SAM-P1/SkuSlc、10周龄、雄)购入作为SAM研究会指定的饲养员的Japan SLC,Inc.生产的小鼠。饵使用自Sankyo Labo Service Corporation,Inc.购入的γ射线灭菌过的固体饲料500N。对于SAM小鼠,在SPF专用动物室中,使用23±2℃、湿度50±10%的恒温·恒湿设备,在铺有灭菌垫料的CLEA Japan,Inc.制的中型聚碳酸酯制使用笼中分别进行饲养。照明在20:00~08:00之间关闭。另外为了比较肿瘤形成,对于正常型的SAM-R1/SkuSlc也同样地进行饲养。
将制造例1的植物发酵物用离子交换水稀释,制成2%和0.2%水溶液,以饮料水的形式使小鼠自由摄取。饲料使用自Sankyo Labo Service Corporation,Inc.购入的γ射线灭菌饲料N500。各组使用8只,饲养当初设为4只/笼,依次移至个别饲养。正常型SAM-R1使用4只,不给予植物发酵物,使其仅自由摄取离子交换水。移至个别饲养后,每周实施1次的评价·观察和笼交换。一般的观测项目设为体重、饮水量(=样品摄取量)、摄食量、外观。老化指标的观察使用SAM研究会指定的评价说明书中记载的标准的评价方式,记载用纸使用SAM研究会指定的片。进而,虽然SAM研究会的评价方式中未包含,但是还实施了存活率的推移、能够自体表观察到的肿瘤的大小测量。
SAM研究会指定的老化指标为以下的方法。
行为(Behavious)(2项目)
1.反应(Reactivity)(级别0-4)
小鼠出至笼外时可见的探索行动
0:低龄小鼠显示出的动作
1:蹑足而行(仓皇逃窜)或者兴奋状态
2:缓慢行走(慢吞吞地走)
3:不动,但触摸臀部时移动
4:完全不动
2.被动性(Passivity)(级别0-4)
从正上偷偷地按压小鼠的脖子时的逃避行动
0:低龄小鼠显示出的动作。强力地猛烈地逃避。
1:逃避慢
2:无逃避
3:无逃避。抓住脖子而向上仰时,翻身
4:即使抓住前肢、后肢也没有挣扎。
光泽外观(Gross Appearances)(9项目)
皮肤和毛发(4项目)
3.光泽度(Glossiness)(级别0-4)
毛的光泽
0:为纯白而闪亮(低龄小鼠显示的毛光泽)
1:0与1之间
2:光泽清晰地消失,没有被污染。
3:2与4之间
4:被严重污染的状态
4.粗糙程度(Coarseness)(级别0-4)
用面积判断用指尖摩擦毛皮时感到的坚硬的触感。
有时有小的溃疡、皮肤炎
0:无毛疙瘩。平滑、光滑
1:位于头、脸附近
2:扩展至肩附近。
3:扩展至背附近。
4:超过背直至尾巴附近。
5.毛发的流失(Loss of Hair)(级别0-4)
毛皮变薄的状态或者光秃的状态。腹部的毛薄因此在背部观察。妊娠小鼠的毛变薄,因此可以观察清楚的光秃。
0:完全没有毛的损失
1:A;光秃为头部程度的大小。
B;毛薄的部分直至背部整体的约一半。
2:A;光秃为背部整体的1/4以下
B;毛薄的部分为背部整体的1/2以上
3:光秃为背部整体的大体1/4以上且1/2以下
4:光秃为背部整体的1/2以上
6.皮肤溃疡(Skin Ulcers)(级别0-4)
也包含白的瘢痕。查找身体整体。
0:完全没有
1:伴随着瘢痕(皮肤白且肥厚)、痂皮的溃疡。无论大小如何。
2:渗出地红色溃烂的溃疡。大体头部的面积左右以下。
3:渗出地红色溃烂的溃疡。大体头部的面积左右以上且背部的面积的1/2程度以下。
4:渗出地红色溃烂的溃疡。大体背部的面积的1/2左右以上。
眼睛(4项目)
左右分别进行评价。总分中直接进行加和。也可以根据需要带有1/2的系数,或仅采用容易评价的项目。角膜病变、白内障的观察直接使用笔灯。
7.眼周病变(Periophthalmic Lesions)(级别0-3)
0:无变化。眼张开。
1:眼睑肿胀而眼合上。或者仅眼的周围有损害。
2:眼周围的损害扩展至鼻尖。
眼没有问题,仅有鼻尖的溃疡,因此应注意。
3:损害扩展至脸整体的状态
8.角膜浑浊(Coreneal Opacity)(级别0-3)
角膜的混浊。角膜白而浑浊,但表面平滑。
放入冰箱,变白的“馒头碎屑”那样的状态。
0:无病变
1:可见虹彩
2:未见虹彩,但可见来自视网膜的反射(黄金色-橙色)
3:无反射光。
9.角膜溃疡(Ulcers of Cornea)(级别0-3)
角膜白浊,表面粗糙。
0:无病变
1:眼裂部一致处有线状的白浊。
2:1与3之间。有来自周围的反射光。
3:基本扩展至角膜整体,未见视网膜的反射光。
10.白内障(Cataract)(级别0-2)
白内障。白浊通过透明的角膜在底部投影出白色。从侧方观察时,可知角膜为透明。
0:无白浊。视网膜反射光正常
1:反射光稍减弱。
2:完全没有反射。(未观察到黄金-橙色)
脊椎(Spine)(1项目)
11.脊椎弯曲(Lordokyphosis)(级别0-3)
抚摸时,卡在头部。
设置小鼠的姿势使其头部伸展。
0:无卡住。
1:轻轻地抚摸时卡住,但强力地抚摸时不会卡住。
2:即使强力地抚摸也卡住。拉伸尾巴伸展脊梁时,不会卡住。
3:无论怎么样都卡住。
总分(Total Score)
全部单纯地加和,作为小鼠个体的老化度评分。眼睑闭合时,将生理盐水取至注射器,清洗眼时使得能够睁开眼,侵袭稍变强。角膜病变强而无法观察到白内障时,严格地讲,该小鼠不得不被省略。
依据SAM研究会的上述老化度评价法,每周评价各小鼠1次,记入研究会指定的记录用纸。同时进行体重测定、饮水量(样品摄取量)的记录、摄食量的记录。SAM研究会的指定评价法中不包含,但记录存活的记录与存活曲线,另外60周龄及其以后,根据鼠颈部中频发的淋巴癌样的肿瘤的长径、短径、高度的测量记录肿瘤体积(mm3)。
图4中示出存活曲线,图5中示出存活率的Kaplan-Meyer解析,表1中示出植物发酵物与CoQ10和CoQ10还原型三者的存活率的平均天数的比较表,表2中示出存活期间的平均值、中央值、最长成长期间,图6中示出老化综合评分的推移,图7中示出54周以及60周龄的老化综合评分的显著性差异解析,图8中示出54周龄以及60周龄的背骨弯曲的比较。另外图9中示出超过60周龄而饲养时频发的肿瘤的体积的推移比较。
由图4表明,植物发酵物2%组与植物发酵物0.2%组相比,存活率高,有浓度依赖性。另外,由图5表明,根据两者间的显著性检验的结果,算出Cox-Mantel-Henzel的显著性检验的P值=0.00461,植物发酵物2%组显著具有延命效果。
对于Kaplan-Meyer解析(图5)中算出的平均周龄(和平均天数),与使用同一小鼠种(SAM-P1♂)的基于CoQ10和CoQ10还原型的试验结果(非专利文献14)进行比较(表1)。植物发酵物2%的平均天数(624.475天)与CoQ10(517.89天)和CoQ10还原型(519.64天)相比,超过误差范围,为较长。需要说明的是,报道了CoQ10与CoQ10还原型之间没有显著性差异。
[表1]
[表2]
如图6所示那样,植物发酵物2%组与0.2%组相比,老化度的体现延迟,另外老化综合分数与0.2%组相比,也始终为低值。
如图6所示那样,全部周龄中,对于植物发酵物2%组,老化指标综合分中,低于0.2%组,对于进行了显著性检验的54周龄、60周龄,2%组与0.2%组相比,在t-检验的P<0.05下老化分数均显著低(图7)。
对于54周龄、60周龄,2%组与0.2%组相比,背骨老化指标分数均显著显示出低值,表明植物发酵物浓度依赖性地抑制伴随着老龄的背骨弯曲异常(图8)。
已知的是,小鼠一般超过60周龄时肿瘤自然发生,大多为淋巴癌。试验例2中,70周龄以上时,正常型(SAM-R1)和老化促进型(SAM-P1)均主要在鼠颈部发生肿瘤,因此,将尺寸以短径x长径x高度计,而近似地测量体积的推移(图9)。需要说明的是,没有实施关于癌种类的细胞诊断,因此假定设为“肿瘤”。老化促进型的植物发酵物2%组与正常型的肿瘤尺寸的推移等同,但老化促进型的植物发酵物0.2%组中,肿瘤尺寸以高于这些组的值推移。
产业上的可利用性
本发明的老化抑制剂具有抑制伴随着动物的老龄的老化症状的发生和存活率延长作用。该老化抑制剂为在抑制老化症状的同时、除了哺乳动物之外对具有动物的基本条件的氧敏感性的线虫也用量依赖性地显示出显著的存活率的延长的首个例子,可以利用为不仅抑制宽范围的老化症状而且也带来存活率延长的新型药物或食品。
Claims (9)
1.一种老化抑制剂,其特征在于,含有植物发酵物作为有效成分,所述植物发酵物为如下(a)~(g)的混合物,
(a)选自由大麦、黑豆、红米、黑米、小豆、薏仁、稗子、小米和黄米组成的组中的1种或2种以上的豆/谷物类的曲霉发酵物;
(b)选自由蜜柑、葡萄、苹果、山葡萄、桃子、柿子、木瓜、梨、西瓜、梅子、无花果、榅桲、南瓜、金橘、柚子、枇杷、杏、枣、栗子、葛枣和李子组成的组中的1种或2种以上的水果类的酵母和/或乳酸菌发酵物;
(c)选自由紫薯、洋姜、胡萝卜、洋葱、甘薯、芋头、薯蓣、萝卜、红芜菁、牛蒡、莲藕、雪莲果、百合根、慈菇、姜、大蒜和姜黄组成的组中的1种或2种以上的块根类/薯类的酵母和/或乳酸菌发酵物;
(d)选自由卷心菜、紫苏、桑叶、鱼腥草、苦艾、山白竹和蒲公英组成的组中的1种或2种以上的花/叶菜类的酵母和/或乳酸菌发酵物;
(e)选自由海带、裙带菜和海蕴组成的组中的1种或2种以上的海藻类的酵母和/或乳酸菌发酵物;
(f)选自由黑芝麻、胡桃和银杏组成的组中的1种或2种以上的种子类的酵母和/或乳酸菌发酵物;
(g)选自由灰树花和香菇组成的组中的1种或2种的蘑菇类的酵母和/或乳酸菌发酵物。
2.根据权利要求1所述的老化抑制剂,其中,植物发酵物含有每100g植物发酵物为以下组成的氨基酸,
3.根据权利要求1或2所述的老化抑制剂,其中,植物发酵物含有每100g植物发酵物为以下组成的有机酸,
4.根据权利要求1~3中任一项所述的老化抑制剂,其中,植物发酵物含有每1g植物发酵物为以下组成的多酚,
5.根据权利要求1~4中任一项所述的老化抑制剂,其中,乳酸菌为选自由乳酸片球菌(P.acidilacti)、短乳杆菌(L.brevis)、肠系膜明串珠菌(L.mesenteroides)、植物乳杆菌(L.plantarum)、乳酸乳球菌(L.lactis)、清酒乳杆菌(L.sakei)、戊糖片球菌(P.pentosaceus)、干酪乳杆菌(L.casei)和弯曲乳杆菌(L.curvatus)组成的组中的1种或2种以上。
6.根据权利要求1~5中任一项所述的老化抑制剂,其中,酵母为酿酒酵母(S.cervisiae)和/或鲁氏酵母(Z.rouxii)。
7.一种饮食品,其含有权利要求1~6中任一项所述的老化抑制剂。
8.根据权利要求1~6中任一项所述的老化抑制剂,其为药品。
9.一种寿命延长剂,其特征在于,含有植物发酵物作为有效成分,所述植物发酵物为如下(a)~(g)的混合物,
(a)选自由大麦、黑豆、红米、黑米、小豆、薏仁、稗子、小米和黄米组成的组中的1种或2种以上的豆/谷物类的曲霉发酵物;
(b)选自由蜜柑、葡萄、苹果、山葡萄、桃子、柿子、木瓜、梨、西瓜、梅子、无花果、榅桲、南瓜、金橘、柚子、枇杷、杏、枣、栗子、葛枣和李子组成的组中的1种或2种以上的水果类的酵母和/或乳酸菌发酵物;
(c)选自由紫薯、洋姜、胡萝卜、洋葱、甘薯、芋头、薯蓣、萝卜、红芜菁、牛蒡、莲藕、雪莲果、百合根、慈菇、姜、大蒜和姜黄组成的组中的1种或2种以上的块根类/薯类的酵母和/或乳酸菌发酵物;
(d)选自由卷心菜、紫苏、桑叶、鱼腥草、苦艾、山白竹和蒲公英组成的组中的1种或2种以上的花/叶菜类的酵母和/或乳酸菌发酵物;
(e)选自由海带、裙带菜和海蕴组成的组中的1种或2种以上的海藻类的酵母和/或乳酸菌发酵物;
(f)选自由黑芝麻、胡桃和银杏组成的组中的1种或2种以上的种子类的酵母和/或乳酸菌发酵物;
(g)选自由灰树花和香菇组成的组中的1种或2种的蘑菇类的酵母和/或乳酸菌发酵物。
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US10226441B2 (en) | 2019-03-12 |
EP3231437B1 (en) | 2020-02-05 |
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EP3231437A4 (en) | 2018-01-17 |
JPWO2016093104A1 (ja) | 2017-04-27 |
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JP6013670B1 (ja) | 2016-10-25 |
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