CN114052259A - 一种具有延缓皮肤衰老作用的组合物及其应用 - Google Patents
一种具有延缓皮肤衰老作用的组合物及其应用 Download PDFInfo
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- CN114052259A CN114052259A CN202111681224.XA CN202111681224A CN114052259A CN 114052259 A CN114052259 A CN 114052259A CN 202111681224 A CN202111681224 A CN 202111681224A CN 114052259 A CN114052259 A CN 114052259A
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- tremella polysaccharide
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Abstract
本发明提供一种具有延缓皮肤衰老作用的组合物及其应用,基于该组合物,可以有效增强胶原蛋白肽延缓皮肤衰老的功效,而且该组合物组成简单,易于规避现有技术中存在的配方组成复杂、质量不可控、功效不稳定、作用机理不明等不足。本发明提供的具有延缓皮肤衰老作用的组合物,活性成分为胶原蛋白肽和银耳多糖,二者重量比为8‑9:1,且所述银耳多糖的平均分子量在3×106Da至4×106Da之间。
Description
技术领域
本发明涉及食品保健领域,特别涉及一种具有延缓皮肤衰老作用的组合物及其应用。
背景技术
随着全球人口老龄化和人们预期平均寿命的增加,衰老引发的健康问题成为全社会共同面临的挑战。从生物学角度看,衰老是生物体内渐进性、结构和功能退行性的生理过程。机体衰老伴随着一系列生理变化,如皮肤胶原蛋白和透明质酸含量下降、氧化应激增加、炎性因子慢性升高、肠道菌群改变等。皮肤作为分隔机体与外界环境的屏障,受内源性(基因组成与表达活性)和外源性(环境、营养和生活方式)致衰因素的双重影响,呈现出肉眼可见的皱纹、松弛、色素沉着、干燥等肌肤衰老特征。拥有“看起来比实际年龄更年轻”的健康皮肤对个人自信心和幸福感至关重要。近年来,胶原蛋白肽作为最常见的食品功能成分之一广泛应用于皮肤美容产品。胶原蛋白肽是从含胶原蛋白丰富的动物皮、骨或鱼皮、鱼鳞经提取、酶解后获得的产物,大量研究表明摄入胶原蛋白肽能帮助皮肤延缓衰老进程。比如在随机双盲安慰剂对照实验中,年龄较大人群(>35岁)补充胶原蛋白肽能减少皮肤皱纹、增加弹性、提高皮肤胶原蛋白原和弹性蛋白含量,使皮肤更健康年轻。随着技术的发展和市场竞争的加剧,美容食品市场上出现了大量的各种类型胶原蛋白肽类产品,组方成分复杂多样,除胶原蛋白肽外还会添加许多其它的成分如果汁、中药/中药提取物、维生素等,以期提高胶原蛋白肽的功效。然而,不同原料配方产品的实际功效可能千差万别,甚至不同成分之间作用可能存在互相抵消,并且复杂的组方往往存在质量(功效成分无法量化)不可控制,导致功效不稳定,同时,复杂的组方制造成本相对也比较高昂。因此,寻找增强胶原蛋白肽延缓皮肤衰老功效的简单有效、机理明确的方法,具有现实的意义。
发明内容
有鉴于此,本发明提供一种组成简单的具有延缓皮肤衰老作用的组合物,基于该组合物,可以有效增强胶原蛋白肽延缓皮肤衰老的功效,而且该组合物组成简单,易于规避现有技术中存在的配方组成复杂、质量不可控、功效不稳定、作用机理不明等不足。
本发明为达到其目的,提供如下技术方案:
本发明提供一种具有延缓皮肤衰老作用的组合物,所述组合物中的活性成分为胶原蛋白肽和银耳多糖,二者重量比为8-9:1,且所述银耳多糖的平均分子量在3×106Da至4×106Da之间。
优选的,所述胶原蛋白肽和银耳多糖二者重量比为8.3:1
优选的,所述银耳多糖的总糖含量为85-90wt%,糖醛酸含量为15-20wt%,且不含蛋白质和三螺旋构象。
优选的,所述银耳多糖平均分子为3.59×106Da,总糖含量为89.5wt%,糖醛酸含量为19.3wt%,且不含蛋白质和三螺旋构象。
本发明还提供一种具有延缓皮肤衰老作用的食品,所述食品中含有上文所述的组合物。
食品的形态可以为但不限于饮料、粉剂或凝胶糖果。
所述食品中可添加有食品领域允许添加的辅料,例如为满足特定的非功效性需求而添加的辅料,例如制备特定形态或特定口味等需求的产品所需的辅料,例如低聚果糖浆、柠檬酸、卡拉胶、魔芋胶、低聚异麦芽糖、罗汉果甜苷等。
本发明还提供上文所述的组合物在制备具有延缓皮肤衰老作用的制剂中应用。
本发明提供的技术方案具有如下有益效果:
经动物试验证明,相比于单一的胶原蛋白肽,本发明提供的胶原蛋白肽和特定的银耳多糖构成的组合物可极其显著地提高衰老皮肤中透明质酸和羟脯氨酸的含量。比以往具有延缓皮肤衰老作用的胶原蛋白肽类组合物相比,配伍简单、成分质量指标清晰可控、功效可保证、作用机理清晰。基于本发明的技术方案,能大幅度提高胶原蛋白肽类功能性食品的质量技术水平。
附图说明
图1为银耳多糖分子量HPGPC图;
图2为银耳多糖溶液的紫外扫描图;
图3为银耳多糖溶液的刚果红染色分析;
图4为实验期间各组小鼠体重的变化;
图5为胶原蛋白肽、胶原蛋白肽复合银耳多糖对小鼠皮肤羟脯氨酸(A)和透明质酸(B)含量的影响;
图6为胶原蛋白肽、胶原蛋白肽复合银耳多糖对小鼠血清、肝脏、心脏部位GSH-Px(A)、SOD(B)活力和MDA(C)浓度的影响;
图7为胶原蛋白肽、胶原蛋白肽复合银耳多糖对IL-1β(A)、TNF-α(B)浓度影响。
具体实施方式
为了便于理解本发明,下面将结合附图对本发明进行更全面的描述。附图中给出了本发明的较佳实施例。但是,本发明可以以许多不同的形式来实现,并不限于本文所描述的实施例。相反地,提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
除非另有定义,本文所使用的所有的技术和科学术语与本发明所属技术领域的技术人员通常理解的含义相同。
以下实施例中使用的主要原料说明如下:
胶原蛋白肽:采购自德国嘉莉达公司,分子量≤10000Da;
麦芽糊精:采购自山东保龄宝公司;
低聚果糖浆:75wt%规格,采购自山东百龙创园公司;
卡拉胶:采购自汕头市捷成生物科技有限公司;
魔芋胶:采购自汕头市捷成生物科技有限公司;
低聚异麦芽糖:90wt%规格,采购自山东百龙创园公司;
罗汉果甜苷:采购自湖南绿蔓生物科技股份有限公司。
对银耳多糖原料进行的结构性质分析说明如下:
以下检测方法均为本领域常规检测方法,未特别说明之处均为本领域技术人员根据所掌握的现有技术或公知常识所能理解或知晓的。
总糖含量采用硫酸苯酚法检测,以甘露糖为对照品;测得银耳多糖总多糖含量为89.5wt%。
分子量采用HPGPC(high-performance gel permeation chromatography,高效凝胶色谱法)方法测定,将银耳多糖用0.02mol/L的KH2PO4缓冲溶液(该溶液也为流动相所用溶液)配制成2mg·mL-1,经0.45μm滤膜过滤后注入色谱***检测:凝胶柱为Ultrahydrogel1000(7.8×300mm)和Ultrahydrogel 500(7.8×300mm)串联使用,流速为0.8mL/min,柱温35℃。结果如图1所示,图1中第一个峰为多糖峰,第二个峰为溶剂峰,银耳多糖的平均分子量Mw为3.59×106Da。
糖醛酸含量采用间羟联苯法,以葡萄糖醛酸为对照品,测得糖醛酸含量为19.3wt%。
银耳多糖用去离子水溶解成1mg/mL溶液,在200~400nm波长下紫外扫描进行检测,结果如图2所示,可见在260nm和280nm处没有明显的吸收峰,说明样品中不含核酸和蛋白质类物质。
银耳多糖在溶液中的构象采用刚果红法检测,银耳多糖用去离子水溶解成1mg/mL溶液,后加入2.0mL 100μmol/L刚果红试剂摇匀,逐渐加入4M的NaOH,混合10分钟后测定。采用酵母β-葡聚糖(Yeastβ-glucan)作为阳性对照。结果参见图3,可见,最大吸收波长随着NaOH浓度的增加而缓慢下降,无吸收波长的增加现象,而阳性对照(Yeastβ-glucan-刚果红溶液)随NaOH浓度的增加,最大吸收波长先增加后下降,表明银耳多糖无三螺旋结构。
实施例1
银耳多糖100g,胶原蛋白肽830g,置于夹层锅中,加入纯化水10L,开启加热至60℃,持续搅拌至银耳多糖和胶原蛋白肽完全溶解,然后加入400g低聚果糖浆,搅拌均匀后,用柠檬酸调pH至4.3-4.5,100目过滤后灌装50ml玻璃瓶,121℃、20min杀菌后得银耳多糖胶原蛋白肽口服液。
实施例2
在溶胶罐中加入80kg纯化水加热至80-90℃,将50g卡拉胶、480魔芋胶和100g银耳多糖投入到溶胶罐中搅拌30分钟至完全溶解,然后转移至配制罐中。将830g胶原蛋白肽、7kg低聚异麦芽糖、100g罗汉果甜苷用50kg 85℃热水溶解后再投入到配制罐中与前述溶液混合,继续搅拌10min。用柠檬酸将pH范围控制在4.4-4.6。然后将溶液80目过滤,然后罐装和巴氏杀菌:罐装体积30g±2.5g,杀菌参数设定为85℃,30分钟。待冷却干燥、包装得银耳多糖胶原蛋白肽果冻。
实施例3
银耳多糖100g,胶原蛋白肽830g,麦芽糊精1060g,、罗汉果甜苷10g,四种物料混合均匀,然后用粉剂包装机包装成2g/包的成品,得银耳多糖胶原蛋白肽粉剂。
银耳多糖促胶原蛋白肽增强延缓皮肤衰老的功效验证
1、动物实验方法
动物(小鼠)饲养于SPF级动物房(环境符合GB14925-2010屏障环境技术要求),实验场所为广州中医药大学实验动物中心(许可证编号为SYXK(粤)2018-0085)。实验伦理由广州中医药大学实验动物伦理委员会审查批准。
动物自由摄食饮水。动物适应两周后按体重随机分组:正常对照组(下文或称为Normal)、模型对照组(下文或称为Model)、胶原蛋白肽组(Collagen peptide,下文或称为CP:830mg/kg·day)、胶原蛋白肽复合银耳多糖组(Complex of collagen peptide andTFPs,下文或称为CPTP:胶原蛋白肽830mg/kg·day,银耳多糖100mg/kg·day),每组8只。
整个实验过程中,正常对照组每天腹腔注射生理盐水,其余各组每天腹腔注射D-半乳糖120mg/kg,同时,实验组每天灌胃上述剂量样品(按照0.1ml/10g体积灌胃),模型组及正常对照组每天灌胃同体积生理盐水,连续8周。末次给药后,小鼠禁食不禁水。第二天,麻醉后眼眶静脉丛取血,4℃、3500r/min离心分离血清中按试剂盒(丙二醛(MDA)试剂盒、总超氧化物歧化酶(SOD)测定试剂盒、谷胱甘肽过氧化物酶(GSH-Px)试剂盒,采购自南京建成生物科技有限公司;小鼠IL-1βELISA试剂盒、小鼠TNF-αELISA试剂盒,采购自江苏酶免实业有限公司)操作说明测定血清中SOD、GSH-Px活性和MDA、IL-1β、TNF-α浓度。
组织中SOD,GSH-Px酶活和MDA含量的测定:无菌快速取小鼠心、肝组织用预冷PBS漂洗去除血液,加PBS制备成10%匀浆液,离心后取上清。先用BCA试剂盒(BCA蛋白定量试剂盒,采购自北京康为世纪生物科技有限公司)进行蛋白定量,再分别按试剂盒说明测定各组SOD、GSH-Px活性和MDA含量。
皮肤中羟脯氨酸、透明质酸含量的测定:取各组小鼠背部脱毛后皮肤约1cm2,用丙酮-***(1:1)(V/V)脱脂,切碎晾干后精密称取20mg,按试剂盒(小鼠透明质酸(HA)ELISA试剂盒、小鼠羟脯氨酸(Hyp)ELISA试剂盒,采购自江苏酶免实业有限公司)操作说明测定羟脯氨酸、透明质酸含量。
2、数据处理所有数据均以Mean±SD形式表示,SPSS 20.0软件进行统计分析。各组进行组间比较之前先进行正态分布和方差齐性检验,组间比较采用单因素方差分析(one-way ANOVA)进行统计检验,P<0.05代表有显著性差异,P<0.01代表极显著差异。
3、结果
3.1对小鼠体征的影响
在实验期间,各组小鼠摄食正常,Normal组皮毛有光泽、行动活跃;Model组皮毛无光泽、活动减少、有红肿现象,表现出衰老特征;CP和CPTP组小鼠皮毛光泽、行动活跃程度相比Model组小鼠有改善。在八周实验期间,各组小鼠体重缓慢增加,组间体重无明显差别(P>0.05),表明造模药物和样品对小鼠生理机能无明显副作用,参见图4,图4中,Normal代表正常组,Model代表模型对照组,CP代表胶原蛋白肽组,CPTP代表胶原蛋白肽复合银耳多糖组。
3.2对小鼠皮肤中羟脯氨酸和透明质酸含量影响
羟脯氨酸是胶原蛋白的特征性氨基酸,皮肤组织中羟脯氨酸的含量反映了皮肤胶原蛋白的含量。如图5所示(其中图A对应羟脯氨酸的含量结果,图B对应透明质酸的含量结果),D-半乳糖注射导致Model组小鼠皮肤中羟脯氨酸和透明质酸含量显著下降(P<0.01),造成皮肤衰老状态;通过8周灌胃,胶原蛋白肽、胶原蛋白肽复合银耳多糖可以显著提升衰老皮肤中羟脯氨酸和透明质酸含量(CP vs.Model,CPTP vs.Model,P<0.01)。特别地,相比于单一的胶原蛋白肽,胶原蛋白肽复合银耳多糖后对衰老皮肤中羟脯氨酸和透明质酸的改善作用也大大增强(羟脯氨酸:7.55±0.18vs.8.86±0.22μg/mgprot;透明质酸:143.36±2.66vs.166.35±1.99ng/mgprot,P<0.01)(图5)。
3.3对体内GSH-Px、SOD活力和MDA含量的影响
结果参见图6,图6中(A)所对应的第一行的三个附图均代表GSH-Px的检测结果,(B)对应的第二行的三个附图均代表SOD的检测结果,(C)对应的第三行的三个附图均代表MDA的检测结果。
SOD、GSH-Px是机体组织中广泛分布的抗氧化酶,酶活力反映机体抗氧化能力的大小。MDA是脂质过氧化产物,MDA含量与机体抗氧化能力负相关。D-半乳糖处理后,与Normal组相比,Model组小鼠血清(Serum)、肝脏(Liver)、心脏(Heart)部位的GSH-Px和SOD活力显著下降,而MDA浓度显著上升(P<0.01)(图6),表明衰老小鼠造模成功。胶原蛋白肽(CP)、胶原蛋白肽复合银耳多糖(CPTP)则可以明显提高衰老小鼠体内GSH-Px活力(P<0.01),但两者提高程度无统计学差异(图6A,P>0.05)。胶原蛋白肽、胶原蛋白肽复合银耳多糖能提高衰老小鼠各组织SOD活力(P<0.01);然而,在肝脏、心脏部位,与单一胶原蛋白肽比较,胶原蛋白肽复合银耳多糖却使SOD活力降低了(肝脏:103.14±4.60vs.85.88±6.85U/mgprot;心脏:137.83±7.00vs.88.54±8.59U/mgprot,P<0.01)(图6B)。相应地,胶原蛋白肽、胶原蛋白肽复合银耳多糖降低了组织中MDA浓度(P<0.01,图6C),与GSH-Px、SOD活力升高作用相吻合;特别地,在肝脏部位,与单一胶原蛋白肽相比,胶原蛋白肽复合银耳多糖进一步降低了MDA浓度(6.82±0.72vs.5.80±0.86nmol/mgprot,P<0.05)。
3.4、对IL-1β和TNF-α分泌影响
结果参见图7,图7中A图对应IL-1β的结果,B图对应TNF-α的结果。
由图7可知,衰老小鼠模型血清中发炎性细胞因子IL-1β、TNF-α极显著升高(P<0.01),表明D-半乳糖处理导致小鼠处于高水平炎症状态。胶原蛋白肽、胶原蛋白肽复合银耳多糖均降低了小鼠血清中IL-1β、TNF-α浓度(P<0.01),说明胶原蛋白肽、胶原蛋白肽复合银耳多糖均具有良好抗炎作用;特别地,胶原蛋白肽复合银耳多糖对炎性细胞因子分泌抑制作用比单一胶原蛋白肽强(IL-1β:479.96±17.37vs.430.95±12.55pg/mL;TNF-α:496.41±19.13vs.427.36±10.12pg/mL,P<0.01)(图7)。机体在炎症状态下,能促进基质金属蛋白酶(Matrix metalloproteinases,MMPs)的表达和活性增加,MMPs是一类调控细胞外基质降解与重塑的重要蛋白酶,能降解细胞外基质的所有成分如胶原、纤维粘连蛋白等。因此,降低衰老模型的炎性因子水平可降低MMPs表达和活性,从而减少胶原蛋白和透明质酸降解延缓皮肤衰老。因而,胶原蛋白肽复合银耳多糖的抗炎作用强于单一胶原蛋白肽,也可能是胶原蛋白肽复合银耳多糖在提高衰老皮肤中羟脯氨酸和透明质酸含量效果优于单一胶原蛋白肽的原因之一。
通过上述试验证明,胶原蛋白肽、胶原蛋白肽复合银耳多糖均可改善D-半乳糖致衰老小鼠的皮肤老化、降低机体氧化压力和炎性细胞因子水平,并且胶原蛋白肽复合银耳多糖对衰老小鼠皮肤中羟脯氨酸和透明质酸含量提升作用优于单一胶原蛋白肽,表明实验样品银耳多糖具有增强胶原蛋白肽延缓皮肤衰老的作用,并且对衰老皮肤的改善作用可能是通过降低氧化应激压力和减少炎性细胞因子水平而实现。
以上所述的仅是本申请的原理和较佳的实施例。应当指出,对于本领域的普通技术人员来说,将分别公开在不同的实施例中的技术方案适当组合而得到的实施方式也包括在本发明的技术范围内,在本申请原理的基础上,还可以做出若干其它变型,也应视为本申请的保护范围。
Claims (8)
1.一种具有延缓皮肤衰老作用的组合物,其特征在于,所述组合物中的活性成分为胶原蛋白肽和银耳多糖,二者重量比为8-9:1,且所述银耳多糖的平均分子量在3×106Da至4×106Da之间。
2.根据权利要求1所述的具有延缓皮肤衰老作用的组合物,其特征在于,胶原蛋白肽和银耳多糖二者重量比为8.3:1。
3.根据权利要求1或2所述的具有延缓皮肤衰老作用的组合物,其特征在于,所述银耳多糖的总糖含量为85-90wt%,糖醛酸含量为15-20wt%,且不含蛋白质和三螺旋构象。
4.根据权利要求3所述的具有延缓皮肤衰老作用的组合物,其特征在于,所述银耳多糖平均分子为3.59×106Da,总糖含量为89.5wt%,糖醛酸含量为19.3wt%,且不含蛋白质和三螺旋构象。
5.一种具有延缓皮肤衰老作用的食品,其特征在于,所述食品中含有权利要求1-4任一项所述的组合物。
6.根据权利要求5所述的具有延缓皮肤衰老作用的食品,其特征在于,食品的形态为饮料、粉剂或凝胶糖果。
7.根据权利要求6所述的具有延缓皮肤衰老作用的食品,其特征在于,所述食品中添加有食品领域允许添加的辅料。
8.权利要求1-4任一项所述的组合物在制备具有延缓皮肤衰老作用的制剂中应用。
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