CN110167573A - 黑树莓提取物、含有黑树莓提取物的脱发抑制剂和饮食品、黑树莓提取物的制造方法和使用了黑树莓提取物的脱发抑制方法 - Google Patents
黑树莓提取物、含有黑树莓提取物的脱发抑制剂和饮食品、黑树莓提取物的制造方法和使用了黑树莓提取物的脱发抑制方法 Download PDFInfo
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Abstract
本发明的黑树莓提取物具有毛乳头细胞的增殖促进作用。
Description
技术领域
本发明涉及黑树莓提取物、含有黑树莓提取物的脱发抑制剂和饮食品、黑树莓提取物的制造方法和使用了黑树莓提取物的脱发抑制方法。
背景技术
对于大部分生物而言,氧是不可欠缺的,被摄入到体内的氧的百分之几变为活性氧,用于防御细菌感染等。但是,为了维持体内稳态,剩余的活性氧需要通过生物体内的抗氧化酶、食品中的外源抗氧化物质除去。
另外,因紫外线、吸烟、废气、大气污染、应激等产生过量的活性氧时,给生物体组织带来氧化性损伤(例如参见非专利文献1)。其结果是,引起以生活方式疾病、老年性疾病为代表的癌症、痴呆等,可以说90%以上的疾病与过量产生的活性氧存在因果关系(例如参见非专利文献2)。
因此,体内的超氧化物歧化酶(SOD,Superoxide dismutase)的活化以及食品中的抗氧化物质受到关注,建立了由美国农务部(USDA)开发的抗氧化能力的测定方法(ORAC;Oxygen Radical Absorbance Capacity,氧自由基吸收能力),其正在普及(例如参见非专利文献3)。另外,在ORAC值高的食品、即抗氧化能力高的食品最佳100中存在12个品种的莓类食品(例如参见非专利文献4)。
随着年龄增长发生脱发而毛发减少,对于脱发和毛乳头细胞的增殖机理进行了大量分析。明确了脱发现象是由毛囊干细胞的17型胶原蛋白的分解导致的毛囊微缩化引起的(例如参见非专利文献5)。另外认为,如果作为具有胶原蛋白特异性保护作用的热激蛋白的Hsp47高表达,则17型胶原蛋白的分解被抑制,结果带来脱发抑制。
作为还原酶的5α-还原酶将睾酮(雄性激素)转化为双氢睾酮(DHT)。在此,DHT是促进男性的毛发的脱落、稀疏化的最强的雄性激素(例如参见非专利文献6)。因此,认为通过抑制5α-还原酶的酶活性能够防止脱发。
现有技术文献
非专利文献
非专利文献1:Oberdanner,C.ROS and Antioxidant Systems in Apoptosis.VDMVerlag,2008.ISBN 978-3836482110
非专利文献2:Slavin JL et al.Health benefits of fruits andvegetables.dv Nutr.2012.1;3(4):506-516
非专利文献3:Cao G et al.Oxygen-radical absorbance capacity assay forantioxidants.Free Radical Biol.Med.1993.14:303-311
非专利文献4:“ORAC值高的(抗氧化作用高的)食品最佳100名单”[网上(online)](2017年6月30日检索)http://www.anteaoxidant.com/orac#top100.html
非专利文献5:Matsumura H,Mohri Y,Binh NT,Morinaga H,Fukuda M,Ito M,Kurata S,Hoeijmakers J,Nishimura EK.Hair follicle aging is driven bytransepidermal elimination of stem cells via COL17A1 proteolysis.2016.5;351.
非专利文献6:Ulrike Blume-Peytavi;David A.Whiting;Ralph M.Trueb).HairGrowth and Disorders.Springer Science&Business Media.(2008)p368-370.
发明内容
发明所要解决的问题
本发明的目的在于,提供具有能够抑制毛发的脱落、稀疏化的作用的莓类的提取物、含有该提取物的脱发抑制剂和饮食品、莓类的提取物的制造方法。另外,本发明的目的在于,提供使用了莓类的提取物的脱发抑制方法。
本发明人着眼于作为具有抗氧化能力的食品的莓类食品,对莓类食品与抑制毛发的脱落的作用的相关性进行了研究,其结果发现黑树莓的提取物达成了上述目的,从而完成了本发明。
用于解决问题的方法
即,根据本发明,可提供:
(1)一种黑树莓提取物,其具有毛乳头细胞的增殖促进作用;
(2)一种黑树莓提取物,其具有抑制毛发的脱落的作用;
(3)一种脱发抑制剂,其含有(1)或(2)所述的黑树莓提取物;
(4)一种饮食品,其含有(1)或(2)所述的黑树莓提取物;
(5)一种黑树莓提取物的制造方法,其是(1)或(2)所述的黑树莓提取物的制造方法,其包括:对黑树莓的果实进行冷冻的冷冻工序;对上述黑树莓的果实进行解冻的解冻工序;向利用上述解冻工序解冻后的上述黑树莓的果实中添加溶剂并进行搅拌的搅拌工序;和将通过上述搅拌工序搅拌后的上述黑树莓的果实和溶剂静置、进行提取的提取工序;
(6)一种脱发抑制方法,其中,使用了(1)或(2)所述的黑树莓提取物。
发明效果
根据本发明,可以提供具有能够抑制毛发的脱落的作用的、具有能够抑制毛发的脱落、稀疏化的作用的莓类的提取物、含有该提取物的脱发抑制剂和饮食品、莓类的提取物的制造方法。另外,根据本发明,可以提供使用了莓类的提取物的脱发抑制方法。
附图说明
图1是示出毛乳头细胞的增殖促进效果的研究结果的图。
图2是示出毛乳头细胞中的Hsp47和17型胶原蛋白m-RNA水平的测定结果的图。
图3是示出5α-还原酶抑制活性的研究结果的图。
具体实施方式
以下,对本发明的黑树莓提取物进行说明。本发明的黑树莓提取物具有促进毛乳头细胞的增殖的作用。另外,本发明的黑树莓提取物具有抑制毛发的脱落的作用。
作为黑树莓提取物的原料的黑树莓属于蔷薇科悬钩子属。另外,本发明的黑树莓提取物优选使用主要来自黑树莓的果实的提取物。
从黑树莓的果实中提取黑树莓提取物的方法没有特别限定,可以使用溶剂提取、超临界提取等。
作为进行溶剂提取时所使用的提取溶剂,没有特别限定,可以使用水、有机溶剂和它们的混合溶剂。作为有机溶剂,可以使用:甲醇、乙醇、正丙醇、异丙醇、正丁醇等醇类;乙二醇、丙二醇等二醇类;乙酸乙酯;丙酮等。这些有机溶剂可以单独使用一种,也可以组合使用两种以上。另外,可以将水等水系溶剂和有机溶剂混合使用。
其中,优选乙醇、将乙醇与水混合而成的乙醇水溶液,乙醇水溶液中的乙醇的浓度优选为1~80v/v%(EtOH),更优选为10~60v/v%(EtOH),进一步优选为20~40v/v%(EtOH)。
需要说明的是,进行超临界提取的情况下,优选使用制成超临界状态的二氧化碳。
另外,作为进行溶剂提取时的提取方法,可以使用搅拌提取、浸渍提取、振荡提取、回流提取、超声波提取等。其中,优选搅拌提取。
搅拌提取的方法没有特别限定,可以进行使用搅拌器的搅拌、使用捣碎器的搅拌等。
进行搅拌提取时,一边优选以500~20000rpm、更优选以1000~10000rpm、进一步优选以6000~10000rpm的搅拌速度进行搅拌,一边进行优选为几分钟~60分钟、更优选为30分钟~60分钟的提取。需要说明的是,优选在搅拌后静置、进行静置提取。更具体而言,优选使用均质器以6000~10000rpm进行30分钟~60分钟均质化、然后静置24小时进行提取。需要说明的是,通过进行静置、提取,可以进行固液分离。
另外,进行溶剂提取时的温度可以为室温,也可以为加热条件下。另外,进行溶剂提取时的压力可以为常压,也可以为加压下。
另外,通过溶剂提取得到的提取液可以根据需要进行过滤等处理。
另外,可以使通过溶剂提取得到的提取液干燥而制成粉末。作为干燥方法,可以使用公知的方法,可以使用喷雾干燥、真空干燥、冷冻干燥等方法。
需要说明的是,作为黑树莓提取物,可以使用制成液体或粉末状的市售品或现有产品。
本发明的黑树莓提取物可以配合在食品、饮料等中。作为食品,可以列举面包类、面类、点心类、食用肉加工品、鱼类加工品、冷冻食品、果冻类、冰淇淋类、乳制品、各种调料等。另外,除了一般食品以外,也可以配合在特定保健用食品、准药品、健康食品、补充剂中。作为饮料,可以列举清凉饮料水、乳饮料、酒类、茶、红茶饮料、咖啡、果汁饮料、碳酸饮料、矿泉水类、果蔬饮料等。
另外,可以将配合有本发明的黑树莓提取物的食品、饮料制成与片剂、胶囊剂、糖浆等口服给药制剂同样的形态。
在制造配合有本发明的黑树莓提取物的食品、饮料时,可以在不妨碍本发明效果的范围内根据需要添加甜味剂、着色剂、防腐剂、增稠剂、稳定剂、胶凝剂、抗氧化剂、显色剂、漂白剂、乳化剂、膨胀剂、酸味剂、光泽剂、香料等添加剂、溶剂、油。这些添加剂可以单独使用一种,也可以组合使用两种以上。
配合在上述食品、饮料中的黑树莓提取物的比例可以根据使用目的适当调整,配合在上述食品、饮料中的黑树莓提取物的比例优选为0.0001~80重量%,更优选为0.003~50重量%,进一步优选为0.005~30重量%。
本发明的黑树莓提取物对于抑制毛发的脱落是有用的。
实施例
以下,列举实施例对本发明进行说明,但本发明并不限于此。需要说明的是,只要没有特别说明,则本实施例中的份和%为重量基准。在实施例和比较例中,进行了基于ORAC值的抗氧化作用的评价、关于毛乳头细胞的增殖促进效果、毛乳头细胞中的Hsp47和17型胶原蛋白的m-RNA水平的测定和5α还原酶的酶活性抑制效果的研究。
关于试样
(试样的准备)作为试样,使用三种莓类试样(黑树莓(蔷薇科悬钩子属)(以下有时称为“BR”)、蓝莓(杜鹃花科越橘属)(以下有时称为“BB”)和树莓(蔷薇科悬钩子属)(以下有时称为“RB”))的提取物以及作为配合有黑树莓的提取物的粉末制剂的Blabina(注册商标)。
(提取液的制备)
黑树莓、蓝莓和树莓的提取液通过以下方法得到。
首先,将黑树莓、蓝莓和树莓的果实分别在冷冻状态下保存。接着,将各果实解冻并切割。分别称量(约100g)黑树莓、蓝莓和树莓,添加5倍量的30v/v%(EtOH)乙醇水溶液(约500mL),使用捣碎器以8000rpm、30分钟的条件进行搅拌提取。进而,在室温下静置24小时,由此进行静置提取,以150目进行过滤而分别得到上述黑树莓、蓝莓和树莓的乙醇提取液。后述的ORAC的测定在乙醇溶解状态下进行,因此,使用乙醇提取液进行测定。
需要说明的是,关于Blabina,按照使Blabina达到9mg/mL的方式使其溶解于30v/v%(EtOH)中。
(提取物的制备)
使用蒸发器分别从通过上述提取液的制备得到的黑树莓、蓝莓和树莓各自的乙醇提取液、Blabina的乙醇溶液中蒸发除去乙醇成分,得到提取物。需要说明的是,黑树莓、蓝莓和树莓的乙醇提取液含有9mg/mL的提取物(extract)。
测定和评价
(基于ORAC值的抗氧化作用的评价)
测定上述得到的三种莓类样本(黑树莓、蓝莓和树莓的乙醇提取液)的ORAC值。将结果示于表1中。需要说明的是,对于Blabina,没有进行ORAC值的测定。
[表1]
样本的种类 | ORAC值(μMTE/g) |
BR | 62 |
BB | 66 |
RB | 49 |
与一般的抗氧化食品相比,三种莓类样本的ORAC值均显示出了高的值(对于一般的抗氧化食品的ORAC值,参见上述非专利文献4)。三种莓类样本中,蓝莓显示出最高的值,但这是使用乙醇提取液测定的结果,因此,不能涉及到水溶性成分。
另外,约0.1%以上的乙醇对细胞有影响,因此,在进行使用了细胞的测定和评价的情况下,利用使用了30体积%乙醇水溶液的乙醇提取液并不合适。因此,在以下的使用了毛乳头细胞的研究中,使用了通过蒸发器分别从乙醇提取液中蒸发除去乙醇成分后的样本(参见上述“提取物的制备”)。
(毛乳头细胞的增殖促进效果)
作为实验中使用的毛乳头细胞,使用人毛乳头细胞。需要说明的是,作为人毛乳头细胞,使用人毛囊毛乳头细胞(Human Follicle Dermal Papilla Cells)、C-12071(购自PromoCell公司),使用专用专用培养基(毛囊毛乳头细胞培养基补充包(Follicle DermalPapilla Cell Growth Medium Supplement Pack);C-39620,购自PromoCell公司),在37℃、5%CO2的条件下进行培养。在即将实验前利用0.25%胰蛋白酶(Trypsin)/EDTA溶液回收细胞,进行台盼蓝染色处理后,利用自动细胞计数仪Countess(Invitrogen)算出细胞数。然后,接种于24孔板或96孔板,进行24小时预培养后用于实验。
另外,将在上述提取物的制备中得到的三种莓类(黑树莓、蓝莓和树莓)提取物、Blabina的除去乙醇后的提取物分别溶解于超纯水中而制成约11.5mg/mL,得到样本。接着,将各样本进行连续稀释(从稀释100000倍至稀释10倍),分别添加至毛乳头细胞中。
并且,向毛乳头细胞中分别添加将上述四种样本连续稀释后的物质,利用自动细胞计数仪Countess(Invitrogen)算出培养48小时后的细胞数。将结果示于图1中。四种均发现了如下效果:浓度依赖性地增殖促进毛乳头细胞的效果与对照(Control)相比同等地增强约3%~约20%。
(毛乳头细胞中的Hsp47和17型胶原蛋白的m-RNA水平的测定)
作为实验中使用的毛乳头细胞,使用与在上述毛乳头细胞的增殖促进效果中使用的人毛乳头细胞同样的毛乳头细胞,供于实验前的处理也与在毛乳头细胞的增殖促进效果中进行的处理同样地进行。
将在上述提取物的制备中得到的三种莓类(黑树莓、蓝莓和树莓)提取物、Blabina的除去乙醇后的提取物分别溶解于超纯水中而制成约11.5mg/mL,得到样本。接着,将各样本进行连续稀释(从稀释100000倍到稀释10倍),分别添加至毛乳头细胞中。并且,将样本添加至毛乳头细胞中后,利用定量PCR法测定1小时时的Hsp47和17型胶原蛋白的mRNA水平。
作为定量PCR法的具体步骤如下所示地进行。使用Trizol试剂(ambion)从添加有样本的毛乳头细胞中提取出全部RNA。依据PrimeScript RT Master Mix(Takara)的方法从提取的RNA逆转录为cDNA,利用SYBR Premix EX Taq II(Takara)进行扩增。需要说明的是,作为PCR反应液,使用50μL(25μL SYBR Green Mix(2x),1μL cDNA,2μL primer pair mix(引物对混合物)(5pmol/μL each primer(每种引物)),22μL H2O)的PCR反应液,反应在如下条件下进行:使95℃下反应30秒的循环进行1个循环,进一步使95℃下反应5秒和60℃下反应30秒的循环进行50个循环。通过比较Ct法(ΔΔCt法)进行相对定量。RT-PCR中使用的各种靶和管家基因(GAPDH)的引物示于表2中。
[表2]
另外,将各样本添加至毛乳头细胞中起1小时后的结果示于图2中。
作为DeltaDeltaCt值,将2以上的高值判定为显著差异。需要说明的是,关于BR的稀释倍率10倍的结果,认为由于分散不均或过浓而DeltaDeltaCt值降低。
根据图2所示的结果可知,四种样本均可见与Hsp47连动而浓度依赖性地诱导17型胶原蛋白的表达的倾向,Blabina、BR显示出比其它两种更高的诱导能力。
(5α-还原酶抑制活性)
作为实验中使用的毛乳头细胞,使用与在上述毛乳头细胞的增殖促进效果中使用的人毛乳头细胞同样的毛乳头细胞,供于实验前的处理也与在毛乳头细胞的增殖促进效果中进行的处理同样地进行。
将在上述提取物的制备中得到的三种莓类(黑树莓、蓝莓和树莓)提取物、Blabina的除去乙醇后的提取物分别溶解于超纯水中而制成约11.5mg/mL,得到样本。接着,将各样本进行连续稀释(从稀释100000倍至稀释10倍),分别添加至毛乳头细胞中。
而且,作为从睾酮代谢为双氢睾酮的还原酶的5α-还原酶被抑制时,结果是双氢睾酮的产生被抑制,因此,利用ELISA法测定双氢睾酮(DHT)产生量。方法依据DHT ELISA Kit(E18-220;IMMUNOSPEC公司)。将结果示于图3中。在图3中,将相对于未处理的DHT值140pg/mL的最大抑制值即20pg/mL(BR、稀释10倍添加、1小时值)设定为100%抑制值,以抑制百分比(%of inhibition)来表示。
根据图3发现,BR显示出最强的抑制,Blabina紧随其后,而且发现其效果浓度依赖性地持续至1~12小时。
Claims (6)
1.一种黑树莓提取物,其具有毛乳头细胞的增殖促进作用。
2.一种黑树莓提取物,其具有抑制毛发的脱落的作用。
3.一种脱发抑制剂,其含有权利要求1或2所述的黑树莓提取物。
4.一种饮食品,其含有权利要求1或2所述的黑树莓提取物。
5.一种黑树莓提取物的制造方法,其是权利要求1或2所述的黑树莓提取物的制造方法,其包括:
对黑树莓的果实进行冷冻的冷冻工序;
对所述黑树莓的果实进行解冻的解冻工序;
向利用所述解冻工序解冻后的所述黑树莓的果实中添加溶剂并进行搅拌的搅拌工序;和
将通过所述搅拌工序搅拌后的所述黑树莓的果实和溶剂静置、进行提取的提取工序。
6.一种脱发抑制方法,其中,使用了权利要求1或2所述的黑树莓提取物。
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