CN108852924A - 一种具抗老化、美白、抗过敏及细胞修护功能的红藜萃取物 - Google Patents
一种具抗老化、美白、抗过敏及细胞修护功能的红藜萃取物 Download PDFInfo
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Abstract
本发明是有关于一种具有抗老化、美白、抗过敏及细胞修护功能的红藜萃取物,其是将有效剂量的红藜萃取物投予至皮肤细胞,其具有清除DPPH·及ABTS·+自由基能力、还原力、螯合铁离子及抑制脂质过氧化物效能;抑制酪胺酸酶活性;清除一氧化氮(NO)自由基、抑制脂氧合酶(LOX‑1)活性;抑制玻尿酸酶的能力,提高皮肤玻尿酸含量,促进伤口修复,减少皱纹产生;抑制基质金属蛋白酶(MMPs)活性,降低胶原蛋白降解;促进细胞修复的能力及提高抗过敏的能力等机制作用。
Description
技术领域
本发明是有关于一种红藜萃取的应用,尤其是指以红藜(CFKE)作为化妆品、保养品等皮肤外用品的添加物,以达到抗老化、抗氧化、抗发炎、抑制黑色素、抗皱、抗过敏及细胞修复等机制作用的目的。
背景技术
现代的东方人对于白晳的肤色需求日益提高,并且对于健康也同样的重视,因此如何提出一种健康的淡斑方式对于医药外用品产业实是一发展重点。传统植物针对美容保养,多为一些补气活血、调整体质的温补药物,也有少许单、复方直接使用于皮肤美白、除痘、消炎等用途。但是对于植物萃取物中的有效成分的机制分析与其于医药外用品中的配方设计应用,仍是国内医药外用品产业需积极开发的重点。因此如能开发传统植物成为低剂量、高效能的淡斑产品,将有助于提升医药外用品科技产业。
目前发现到的问题是,现有的美白产品里面,多是借由维生素C(Vitamin C)、对苯二酚(Hydroquinone)、杜鹃花酸(Acelaic acid)和熊果素(Arbutin)来达到淡斑的功效,虽然这些物质对于淡斑的确有显著的功效,然而,有些淡斑剂的作用为直接破坏黑色素细胞,因此会造成细胞的毒杀性。因此抑制黑色素生成与抗氧化成分的安全性与其作用浓度是值得重视的。本发明是萃取出天然植物红藜萃取物,其具有清除DPPH·及ABTS·+自由基能力、还原力、螯合铁离子及抑制脂质过氧化物效能;抑制酪胺酸酶活性;清除一氧化氮(NO)自由基、抑制脂氧合酶lipoxygenase-1(LOX-1)活性。
以往中医对于红藜的认知仅为食用植物,具有抗氧化及抗发炎的功效,未有其美白及抗老化的功能发现,而市售产品所含的美白成分例如:维生素C(Vitamin C)、对苯二酚(Hydroquinone)、杜鹃花酸(Acelaic acid)和熊果素(Arbutin),虽具有显著功效,但是有些美白剂的作用为直接破坏黑色素细胞,因此会造成细胞的毒杀性。
发明人即是鉴于上述现有美白、抗老化的产品组合物于实际实施使用时仍具有多处缺失,本于孜孜不倦的精神,借由其丰富专业知识及多年的实务经验加以改善,并据此研创出本发明。
发明内容
红藜为近年新颖的食用植物之一,其组成约有40%为多酚类化合物,以芸香素(rutin)为主要多酚成分,已有许多文献证实rutin有优越的抗氧化及抗发炎等各项活性。有47.8%甜菜红(betanin)成分,然而关于betanin的相关文献讨论却不多见,因此本发明即以红藜及其化合物betanin进行一系列抗氧化、DNA修复、抑制黑色素生合成及抗发炎活性评估。
本发明主要目的为提供一种红藜萃取物的应用,其是指以红藜(Chenopodiumformosanum Koidz,CFK)水萃及乙醇萃取物作为具有清除自由基能力及抑制酪胺酸酶活性的化妆品添加物;借此,红藜萃取物可作为皮肤外用品以用于预防或缓和个体的皮肤老化现象,达到抗老化、抗氧化、抗发炎、抑制黑色素、抗皱、抗过敏及细胞修护等机制作用。
为了达到上述实施目的,本发明一种红藜萃取物的应用,其是将有效剂量的红藜萃取物投至皮肤细胞,其具清除自由基、抑制脂质过氧化物效能以及抑制脂氧合酶lipoxygenase-1(LOX-1)活性,可提高抗氧化、抑制黑色素生成、抗发炎及细胞修复的效果。
较佳者,该红藜萃取物包含水萃CFKW及乙醇萃取物CFKE。
较佳者,该红藜萃取物具有清除或减少自由基以防止细胞老化的能力。
较佳者,该自由基为DPPH(2,2-diphenyl-1-picrylhydrazyl)自由基或ABTS(2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic acid)自由基。
较佳者,该红藜萃取物是作为美白或抗氧化的化妆材料组成物、食品添加物或医药组成物。
较佳者,该红藜萃取物具有抑制UV照射引起的皮肤发炎反应的能力或作为皮肤消炎剂。
较佳者,该红藜萃取物是作为抗发炎的化妆材料组成物或医药组成物。
较佳者,该红藜萃取物具有调节p53及NER修复系统的相关因子来修复由UV照射引起的DNA的损伤的能力。
较佳者,该红藜萃取物具有抑制细胞外蘑菇酪胺酸酶的活性,进而降低黑色素生成的能力。
较佳者,该红藜萃取物可抑制NO及LOX-1的活性达到抑制发炎反应的目的。
较佳者,该红藜萃取物具有抑制玻尿酸酶的能力,提高皮肤玻尿酸含量,促进伤口修复,减少皱纹产生。
较佳者,该红藜萃取物还具有抑制基质金属蛋白酶(MMPs)活性,降低胶原蛋白降解。
较佳者,该红藜萃取物具有促进细胞修复的能力。
较佳者,该红藜萃取物具有提高抗过敏的能力。
附图说明
图1是本发明细胞毒理性测试图。
图2是本发明清除自由基能力测试图。
图3是本发明美白效能测试图。
图4是本发明抗发炎试验图。
图5是本发明DNA保护试验图。
图6是本发明DNA保护对照图。
图7是本发明抗皱试验图。
图8是本发明抑制玻尿酸酶能力试验图。
图9是本发明细胞的伤口愈合能力试验图。
图10是本发明UVB细胞受损试验图。
图11是本发明抗敏能力试验图。
具体实施方式
本发明的目的及其结构功能上的优点,将依据以下附图所示的结构,配合具体实施例予以说明,以使审查委员能对本发明有更深入且具体的了解。
首先,本发明人由红藜分离制得水萃CFKW、乙醇萃取物CFKE,其中该红藜可选用带壳红藜或不带壳红藜或将分离的壳与红藜分别萃取。举例而言,水萃CFKW可为将红藜与水混合以得到水溶性滤液,接着将水溶性滤液干燥而取得的,而混合温度较佳地为80至90℃;乙醇萃取物CFKE可为将红藜与乙醇混合以得到乙醇溶性滤液,接着将乙醇溶性滤液干燥而取得的。红藜与水的重量比较佳地为1:8至1:10,更佳地为1:9;而红藜与乙醇的重量比较佳地为1:4至1:6,更佳地为1:5。此外,为了让水萃CFKW与乙醇萃取物CFKE能与生物细胞完整接触,水萃CFKW与乙醇萃取物CFKE可以混合溶液形式作用于生物细胞。举例而言,水萃CFKW或乙醇萃取物CFKE可先溶解于溶剂(如二次水、DMSO或PBS)中,再以所得的混合溶液作用于生物细胞。上述材料的水萃CFKW及乙醇萃取物CFKE具有降低因H2O2刺激而引起的HaCaT细胞内氧化压力,提高细胞内glutathione(GSH)含量,及提高抗氧化酵素superoxidedismutase(SOD)、catalase和glutathione peroxidase(GPx)的表现量。
红藜萃取物(水萃CFKW、乙醇萃取物CFKE)具有抑制细胞内酪胺酸酶(tyrosinase)活性及降低黑色素生成量的能力。以免疫荧光试验及西方墨点法试验,红藜萃取物(水萃CFKW、乙醇萃取物CFKE)作用于细胞后,证实具有抑制黑色素生合成途经中接受器MC1R(melanocortin-1receptor)和MITF(microphthalmia-associated transcriptionfactor)的表现,并影响酪胺酸酶、TRP-2(tyrosinase-related proteins-2)和TRP-1(tyrosinase-relatedproteins-1)的表现,进而抑制黑色素生成。
在抗发炎能力试验中,红藜萃取物具有抑制细胞内发炎因子(TNF-α、IL-1β及IL-6)的含量的能力,且具有降低发炎相关路径的p38、IL-6、IL-1β和TNF-α蛋白质表现。动物试验SKH-1小鼠经UVB照射后以免疫组织化学染色法immunohistochemistry(IHC)及西方墨点法试验证实红藜萃取物具有降低发炎相关路径的p38、IL-6、IL-1β和TNF-α蛋白质表现。
在DNA修复及保护能力试验中,红藜萃取物具有调节nucleotideexcision repair(NER)修复系统的相关因子来修复由UV照射引起的DNA的损伤的能力,以及调节p53及NER修复系统的相关因子来修复由UV照射引起的DNA损伤的能力。
借由下述具体实施例,可进一步证明本发明可实际应用的范围,但不意欲以任何形式限制本发明的范围。
实施例1:红藜的水萃取
红藜购自台东县池上乡地方农会。将红藜捣粹后,取100克的捣萃物与900克的水于双磨口圆底烧瓶中混合。接着,置入加热包并于80至90℃下以加热回流方式混合捣萃物与水约3小时。时间到达后,将双磨口圆底烧瓶冷却,再以布式漏斗与抽气泵对混合物过滤,而取得澄清滤液。最后,先经减压浓缩滤液,再于-80℃下冷冻浓缩后的滤液约一天,后置于真空瓶中冷冻干燥以得到最终产物水萃物CFKW(萃取率约7.8%,100克红藜可得到7.8克水萃物CFKW)。须说明的是,进行后续的生物实验前,须先将水萃物CFKW溶解于二次水或DMSO等溶剂中,因此下文所提的浓度或类似表示若无特别界定均指水萃物CFKW的重量相对于水萃物CFKW与溶剂的总体积的浓度。
实施例2:红藜的乙醇萃取
红藜购自台东县池上乡地方农会。将红藜捣粹后,取100克的捣萃物浸泡于500克的95%乙醇达三天。接着,以布式漏斗与抽气泵对混合物过滤,而取得澄清滤液。另外,过滤后得到的滤渣再浸泡于500克的95%乙醇达三天后,以布式漏斗与抽气泵对此处的混合物过滤,而取得此处的澄清滤液。最后,于两次所得的滤液混合后,先经减压浓缩滤液,再于-80℃下冷冻浓缩后的滤液约一天,后置于真空瓶中冷冻干燥以得到最终产物乙醇萃取物CFKE(萃取率约3.2%,100克红藜可得到3.2克乙醇萃取物CFKE)。须说明的是,进行后续的生物实验前,须先将乙醇萃取物CFKE溶解于二次水或DMSO等溶剂中,因此下文所提的浓度或类似表示若无特别界定均指乙醇萃取物CFKE的重量相对于乙醇萃取物CFKE与溶剂的总体积的浓度。
实施例3:细胞毒理性测试
鉴于现有的美白剂或皮肤黑色素抑制剂的作用为会造成细胞毒性,因此本发明人在取得此红藜萃取物后,先进行此化合物的细胞毒杀测试实验,利用100、250和500μg/ml浓度的红藜萃取物作用于HaCaT细胞24小时之后,利用MTT试验测定其存活度。其结果如图1所示,红藜的水萃CFKW及乙醇萃取物CFKE在细胞中不具毒性,与市售常用成分ascorbic acid(AA)相比来得安全。
实施例4:清除自由基能力测试
本实验探讨红藜的水萃CFKW、乙醇萃取物CFKE清除自由基的功效,利用DPPH和ABTS+两种自由基测试其清除能力,其结果如图2所示,乙醇萃取物CFKE在清除DPPH自由基试验中约在45μg/ml时就达到50%的清除率,且乙醇萃取物CFKE清除两种自由基的能力更优于水萃CFKW,其结果显示红藜萃取物对于两种自由基有很好的抑制率。
实施例5:美白效能测试
酪胺酸酶为黑色素生合成途径中重要的酵素,将红藜的水萃CFKW及乙醇萃取物CFKE以不同浓度100、250和500μg/ml进行细胞外蘑菇酪胺酸酶活性试验评估。其结果如图3所示,经过红藜的水萃CFKW及乙醇萃取物CFKE作用后,抑制细胞外蘑菇酪胺酸酶活性随浓度增加而提高,且乙醇萃取物CFKE(EC50为247μg/ml)与水萃CFKW(EC50为260μg/ml)的抑制率相当,证实红藜的水萃CFKW及乙醇萃取物CFKE具有抑制细胞外蘑菇酪胺酸酶的活性。
实施例6:抗发炎试验
NO为主要参与发炎反应的介质之一,而LOX-1是最常见引起发炎反应的酵素,借由此试验评估红藜的水萃CFKW及乙醇萃取物CFKE抗发炎效果。其结果如图4所示,红藜的水萃CFKW及乙醇萃取物CFKE,在清除NO的活性上功效相当,而结果显示水萃CFKW在抑制LOX-1的活性效能上更优于乙醇CFKE,证实红藜的水萃CFKW及乙醇萃取物CFKE能经由抑制NO及LOX-1的活性来达到抑制发炎反应的活性,具有抗发炎的效果。
实施例7:DNA保护试验
紫外线曝晒或生理代谢,均会增加氧化压力造成DNA的损伤。本试验以pUC119DNA以H2O2(1mM)诱导及以UVB(20mJ/cm2)照射,引发DNA损伤,评估红藜的水萃CFKW及乙醇萃取物CFKE是否具有保护DNA避免因氧化压力及UVB照射所造成的DNA损伤。请参照图5,pUC119DNA原为超螺旋体结构(Supercoiled form,SC-form),经H2O2(1mM)诱导及UVB(20mJ/cm2)照射后会造成DNA断裂,形成线型(Linear form,L-form)和开放型(Open form,O-form)的DNA片段。续参照图6,当加入不同浓度的乙醇萃取物CFKE(100、250和500μg/ml)再以H2O2诱导及UVB照射后,结果显示乙醇萃取物CFKE能有效保护DNA,且随浓度的增加保护效果越佳,证实红藜有保护DNA免于因氧化压力及UVB照射所造成的损伤。
实施例8:抗皱试验
胶原蛋白主要存在于皮肤组织中真皮层的结缔组织中,是人体内含量最高的蛋白质,有很强的伸展力且能支撑真皮层整体结构,胶原蛋白也是细胞外基质的主要组成成分,保持皮肤弹性并具有修复的能力,而胶原蛋白会随着老化及环境等因素而流失,使皮肤产生皱纹及骨质疏松等问题。其中基质金属蛋白酶(Matrix metalloproteinases,MMPs)为胶原蛋白分解酵素,MMP-2及MMP-9主要是分解第Ⅱ型胶原蛋白。为调节光老化重要因子,纤维母细胞受UV曝晒会引起基质金属蛋白酶大量表现,进而降解细胞外基质(ECM)使真皮组织的胶原蛋白降解。
本试验将红藜的乙醇萃取物CFKE(100、250和500μg/ml)以不同浓度作用于Hs68细胞中培养72小时后,测定胶原蛋白含量。借此试验评估该红藜的乙醇萃取物CFKE是否具有抑制MMPs的活性。其结果如图7所示,经红藜的乙醇萃取物CFKE作用后,与Control(控制组)相比,胶原蛋白含量随着浓度提高而增加,因此可推知红藜的乙醇萃取物CFKE具有抑制MMP-9及MMP-2的活性,并可以有效地增加纤维母细胞生成胶原蛋白的含量。
实施例9:抑制玻尿酸酶能力试验
玻尿酸存在于人体皮肤组织中的真皮层及结缔组织中,减少皱纹的产生,并具有促进伤口修复的功效,而玻尿酸酶是一种会使玻尿酸降解的酵素,使玻尿酸的结构瓦解,丧失原有功能。借由此试验评估红藜的乙醇萃取物CFKE抑制玻尿酸酶的能力。
将红藜的乙醇萃取物CFKE(500μg/ml)及标准品表没食子儿茶素(Epigallocatechin gallate,EGCG),分别加入玻尿酸酶(3000unit/mL)作用18小时,结果如图8所示,经过红藜的乙醇萃取物CFKE作用后的位置和Control相比颜色明显深了许多,经定量分析出红藜的乙醇萃取物CFKE对玻尿酸酶的抑制率为58%。结果证实,红藜的乙醇萃取物CFKE具有抑制玻尿酸酶的能力。
实施例10:细胞之伤口愈合能力试验
以伤口愈合(wound healing)试验评估CFKE是否具有促进细胞修复的能力。首先以红藜萃取物CFKE(500μg/ml)作用于HaCaT细胞4小时后,抽干培养液,以UVB(50mJ/cm2)照射,再加入CFKE作用,培养4小时后,以显微镜观察细胞移动的变化。
结果如图9所示,Control及单纯照射UVB的细胞没有太大的移动变化,尤其是单纯照射UVB的细胞,从0至48小时几乎没有变化。经红藜萃取物CFKE作用后的细胞在24小时后有明显的向中间移动的趋势,到了48小时几乎完全密合。结果再次证实,红藜萃取物CFKE具有促进细胞修复的能力。
实施例11:UVB细胞受损试验
彗星试验是以胶体电泳并以EtBr进行细胞核的染色,侦测单一细胞中DNA经UVB照射后的损伤程度。以CFKE(500μg/ml)作用于HaCaT细胞4小时后,抽干培养液,以UVB(50mJ/cm2)照射,再加入红藜萃取物CFKE作用,培养4小时后,以显微镜观察细胞的损伤程度。
结果如图10所示,单纯受UVB照射的细胞,DNA受损且有明显的拖尾现象,1000颗细胞中,约有50%颗细胞的DNA有受损的情形。经过CFKE作用过后的细胞,在高浓度500μg/ml作用下,1000颗细胞中,约有10%颗细胞的DNA有受损的情形。借此判断CFKE具有保护DNA免于UVB照射所造成的伤害。
CPD为DNA受UVB照射后所产生的光产物,结果如图10所示,经UVB(50mJ/cm2)照射后的细胞,CPD的荧光表现量增加,而经过CFKE(500μg/ml)作用后,明显降低了UVB光产物的生成,再次证实CFKE具有保护DNA并降低因UVB照射所造成的DNA损伤。
实施例12:抗敏能力试验
请参照图11以卡西霉素A23187或抗原诱导的β-葡萄糖醛酸酶去颗粒试验:以卡西霉素A23187和抗原诱导RBL-2H3肥大细胞去颗粒的程度,是由β-葡萄糖醛酸酶释放试验,经由下列方式改良的方法来测定:接种RBL-2H3肥大细胞(2×104cells/well)在96孔微量盘和RBL-2H3肥大细胞(3×104cells/well)在48孔微量盘中,分别供给卡西霉素A23187诱导实验和抗原诱导实验所用,在细胞中加入各种浓度的样品,静置20小时;使用迪皮质醇(10nM)作为正对照组。在抗原诱导实验中,细胞首先以抗-二硝基苯酚IgE单克隆抗体(anti-dinitrophenol IgE,anti-DNP IgE)(5μg/mL)被动高敏至少2小时,将细胞以预热的台罗德氏缓冲液(135mM氯化钠、5mM氯化钾、1.8mM氯化钙、1.0mM氯化镁、5.6mM葡萄糖、20mM4-羟乙基呱嗪乙磺酸,pH 7.4)彻底冲洗后,各别以台罗德氏缓冲液配制的钙离子载体A23187(卡西霉素,1μM)或抗原二硝基苯-胎牛血清共轭物(100ng/mL)刺激一小时。为测量β-葡萄糖醛酸酶释放的总量,将未受刺激的细胞以0.5%的Triton X-100溶液裂解,或者不进行处理使细胞自发性的释放β-葡萄糖醛酸酶;分装上清液(50μL)与等量的1μM聚氮-乙酰葡萄糖胺(抗原)配制于0.1M柠檬酸盐缓冲液(pH 4.5)中,用作释放β-葡萄糖醛酸酶的基底。在37℃下培养1小时后,加入100μL的终止缓冲液(0.1M Na2/NaHCO3,pH 10.0)猝灭反应。将微量盘分析仪(Multiskan Ascent,Thermo Scientific)设定在405nm波长,测量吸亮度;β-葡萄糖醛酸酶释放的抑制百分比是实验组除以对照值(未处理细胞)的百分比来计算;对二甲基亚砜最大的耐受剂量为0.5%,且所有实验重复三次。
样品诱导的β-葡萄糖醛酸酶直接去颗粒试验:在RBL-2H3肥大细胞中,由样品引发的β-葡萄糖醛酸酶的释放量是以改良过的β-葡萄糖醛酸酶释放实验来决定;简单地说,将RBL-2H3肥大细胞(4×104cells/well)接种于48孔微量盘中,加入样品后静置10小时。将台罗德氏缓冲液加入5.6mM葡萄糖、2mg/mL牛血清白蛋白(BSA)和2mM谷氨酸以制备样品和细胞所用。50μL的上清液转移至96孔微量盘,依照前述方式测量β-葡萄糖醛酸酶的释放量,卡西霉素A23187(1μM)做为正对照组,所有实验进行重复三次以上。
综上所述,本发明证实红藜的水萃CFKW及乙醇萃取物CFKE具有清除活性氧和抑制黑色素生成,并具有皮肤表皮细胞的DNA保护、修复能力以及具有抑制发炎反应的产生,具有发展成为美白、抗氧化及抗发炎的保养品或保健食品添加剂的潜力,并确能借由上述所揭露的实施例,达到所预期的使用功效,且本发明亦未曾公开于申请前,诚已完全符合专利法的规定与要求。故依法提出发明专利的申请,恳请惠予审查,并赐准专利,则实感德便。
惟,上述所揭露的图示及说明,仅为本发明的较佳实施例,并不是限定本发明的保护范围;大凡熟悉该项技术的人员,其所依本发明的特征范畴,所作的其它等效变化或修饰,皆应视为不脱离本发明的设计范畴。
Claims (10)
1.一种红藜萃取物用于制备具有抗老化、美白、抗过敏及细胞修护功能的组合物的用途,所述组合物以有效剂量的红藜萃取物投至皮肤细胞,以清除自由基、抑制脂质过氧化物效能以及抑制脂氧合酶lipoxygenase-1(LOX-1)活性,进而提高抗氧化、抑制黑色素生成、抗发炎及细胞修复。
2.如权利要求1所述的用途,其中所述红藜萃取物包含水萃CFKW及乙醇萃取物CFKE。
3.如权利要求1所述的用途,其中所述红藜萃取物系具有清除或减少自由基以防止细胞老化的能力。
4.如权利要求1所述的用途,其中所述自由基为DPPH自由基或ABTS自由基。
5.如权利要求1所述的用途,其中所述红藜萃取物具有调节p53及NER修复系统的相关因子来修复由UV照射引起的DNA的损伤的能力。
6.如权利要求1所述的用途,其中所述红藜萃取物具有抑制细胞外蘑菇酪胺酸酶的活性,进而降低黑色素生成的能力。
7.如权利要求1所述的用途,其中所述红藜萃取物可抑制NO及LOX-1的活性达到抑制发炎反应。
8.如权利要求1所述的用途,其中所述红藜萃取物具有抑制玻尿酸酶的能力,提高皮肤玻尿酸含量,促进伤口修复,减少皱纹产生。
9.如权利要求1所述的用途,其中所述红藜萃取物还具有抑制基质金属蛋白酶活性,降低胶原蛋白降解的能力。
10.如权利要求1所述的用途,其中所述红藜萃取物具有提高抗过敏的能力。
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WO2021073397A1 (zh) * | 2019-10-17 | 2021-04-22 | 财团法人医药工业技术发展中心 | 制备含去壳台湾红藜萃取物而可治疗非酒精性脂肪肝的医药组合物及其应用 |
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