CN114989321B - 怀山药多糖的制备方法及其在祛斑美白和抗衰老中的应用 - Google Patents
怀山药多糖的制备方法及其在祛斑美白和抗衰老中的应用 Download PDFInfo
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- CN114989321B CN114989321B CN202210626601.8A CN202210626601A CN114989321B CN 114989321 B CN114989321 B CN 114989321B CN 202210626601 A CN202210626601 A CN 202210626601A CN 114989321 B CN114989321 B CN 114989321B
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- polysaccharide
- chinese yam
- yam polysaccharide
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- rhizoma dioscoreae
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Classifications
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Abstract
本发明涉及一种怀山药多糖提取工艺及其在祛斑美白和抗衰老中的应用。本发明通过对酪氨酸酶的抑制实验、DPPH自由基和羟自由基清除实验、对双氧水诱导人永生化表皮细胞氧化损伤的保护实验、抑制黑色素细胞的增殖实验、抑制黑色素细胞中的黑色素合成实验,证实怀山药多糖具有美白、淡斑、抗衰老功效,怀山药多糖可被应用于制备祛斑美白、抗衰老面霜和祛斑美白、抗衰老面膜等护肤品。
Description
技术领域
本发明涉及化妆品领域,涉及植物多糖及在化妆品领域中的应用,具体涉及一种怀山药多糖及其制备方法和祛斑美白、抗衰老用途。
背景技术
皮肤老化的原因包括内源性原因与外源性原因,内源性原因是指人体皮肤随着年龄的增长自然老化,出现细小皱纹、皮肤弹性下降、松弛等状况;外源性原因有许多,环境、水质、空气都有一定的影响,但最主要的原因则是日晒造成的光老化,主要表现为皮肤松弛、粗糙,出现晒斑、色素等沉淀,皮肤暗黄无光泽等。抗氧化成分能够在人体皮肤氧化的过程中起到一定的调节作用,能够有效地起到防止光老化的效果,能够减轻皮肤色素沉淀,改善皮肤炎症的不良反应,阻断由于光老化导致的皮肤损害,促进人体皮肤的新陈代谢,加强人体皮肤的免疫力。
皮肤色调的主要决定因素是皮肤内的黑色素,肤色的深浅取决于细胞合成黑色素的能力。酪氨酸酶、多巴醌互变异构酶和5,6-二羟基吲哚-2-羧酸氧化酶与黑色素的生成有关,其中酪氨酸酶是合成黑色素生物合成的关键限速酶,因此酪氨酸酶可作为祛斑美白药物的筛选靶点。目前市面上许多美白产品是通过添加熊果苷的方式来抑制酪氨酸酶活性,但熊果苷具有高度的光敏性,因而需要添加大量的防晒剂,容易对皮肤造成负担加速老化。烟酰胺是另一种常用的美白添加成分,能够抑制黑素小体的转运,但烟酰胺中常因含有杂质烟酸对皮肤造成刺激。
多糖作为天然产物,来源广泛而且大部分价格低廉,与化学合成药物相比,多糖因具有多样的生物学活性以及安全无毒、药效稳定等特点,具有广阔的发展前景。多糖在保湿、促进创伤愈合、治疗特应性皮炎、防紫外线损伤、治疗痤疮、延缓衰老、美白及皮肤屏障损伤修复上具有广泛的应用。
山药为传统的药食两用中药,来源于薯蓣科薯蓣(Dioscorea opposita Thunb.)的干燥根茎。山药性平、味甘,具有补脾养胃、生津益肺、补肾涩精等作用,现代医学研究发现,山药中的活性物质包括多糖、皂苷、脂类、黄酮、蛋白质与氨基酸等成分,其中山药多糖是其主要活性成分之一。近些年来,国内外学者围绕山药多糖的生物活性做了大量研究工作,发现山药多糖在降血糖血脂、保肝护肝、抗肿瘤、增强免疫力、抗氧化、抗衰老、神经保护、调节肠道菌群等方面表现出良好的药理活性。
怀山药多糖是一种高生物活性的天然多糖,具有良好的祛斑美白、抗衰老的生物活性。目前,尚未见怀山药多糖在祛斑美白、抗衰老的领域的研究应用报道。
发明内容
本发明设计了一种怀山药多糖的制备方法及其在祛斑美白和抗衰老中的应用,其解决的技术问题是现有技术中尚未见怀山药多糖在祛斑美白、抗衰老的领域的应用。
为了解决上述存在的技术问题,本发明采用了以下方案:
一种怀山药多糖的制备方法,包括以下步骤:
步骤1、怀山药烘干粉碎;
步骤2、微波提取怀山药多糖水提液;
步骤3、怀山药多糖水提取液浓缩;
步骤4、浓缩液的醇沉并收集沉淀物;
步骤5、沉淀物除蛋白,并收集下相溶液;
步骤6、透析;
步骤7、冷冻干燥即得怀山药多糖。
优选地,步骤1中怀山药根茎部分洗净,在60℃烘箱烘干至恒重,粉碎机粉碎后过20目筛;
优选地,步骤2中筛选后山药粉末和水按料液比1:25混合,80℃微波提取20分钟;将怀山药多糖提取液离心,所得滤渣再次重复提取1次,合并滤液,即为怀山药多糖水提取液。
优选地,步骤3中将怀山药多糖水提取液减压浓缩至原体积的五分之一;
或者,步骤4中向怀山药多糖浓缩液中加入适量95%乙醇使其乙醇终浓度为80%,4℃放置过夜,可见白色沉淀析出,8000r/min离心5min,收集多糖沉淀物。
优选地,步骤5中将多糖沉淀物复溶于水,水溶液与乙醇、硫酸铵按照质量比5.159:2.811:2.03的比例配置成乙醇/硫酸铵双水相体系,倒入分液漏斗中,静止过夜,随后收集下相溶液;
优选地,步骤6中将下相溶液放入截留分子量为1000Da的透析袋中,先用自来水透析24h,随后在去离子水中透析24h,每4h换水一次;
优选地,步骤7中将透析液减压浓缩后,冷冻干燥后即得怀山药多糖;按照苯酚-硫酸法测定多糖含量,每100g山药粉可得9.6g多糖;凝胶色谱排阻法测得分子量分布为5kDa-500kDa,优选为10.3kDa-131.5 kDa。
一种怀山药多糖,其特征在于:使用上述制备方法制得,所述怀山药多糖具有抑制酪氨酸酶活性或抗氧化活性。
上述怀山药多糖对DPPH自由基清除的应用。
上述怀山药多糖对羟自由基清除的应用。
上述怀山药多糖对酪氨酸酶抑制的应用。
上述怀山药多糖对双氧水诱导人永生化表皮细胞氧化损伤有保护作用的应用。
上述怀山药多糖可抑制黑色素细胞的增殖或抑制黑色素细胞中的黑色素合成的应用。
上述怀山药多糖在制备具有美白功效的药品或保健品肤外用剂的应用。
上述怀山药多糖在制备具有淡斑功效的药品、保健品或皮肤外用剂的应用。
上述怀山药多糖在制备具有抗衰老功效的药品、保健品或皮肤外用剂的应用。
所述山药多糖在药品或保健品中可与药学可接受的辅料支撑口服或外用剂,如:片剂、胶囊剂、颗粒剂、丸剂,膏剂等。
该怀山药多糖的制备方法及其在祛斑美白和抗衰老中的应用具有以下有益效果:
(1)本发明采用微波法提取怀山药多糖,时间短,提取效率高。双水相萃取法纯化怀山药多糖,与传统的sevage法相比,不存在有机溶剂残留问题,操作条件温和,各种参数按比例放大多糖收率不会降低;双水相体系之间的传质和平衡速度快,分离效率高;易于进行连续化操作。
(2)本发明怀山药多糖具有多种生物活性,虽然已经开发出一些护肝剂、免疫调节等产品,但在化妆品领域的应用几乎空白,而山药多糖具有良好抗氧化、抗衰老活性,具有优越的抗氧化、防止光老化护肤产品的开发潜力。本研究探究其美容功效及开发利用价值,研制山药多糖面膜护肤产品,既适应了绿色环保的理念来满足求美者的需求,又能对农产品进行深加工利用,把植物抗氧化精华运用于植物化妆品中,发挥优势作用。
(3)本发明怀山药多糖具有祛斑、美白、抗氧化、抗衰老等作用,可被用于日化领域,如护肤品和/化妆品;也可用作药品和保健品。
(4)本发明步骤2中使用微波提取多糖的提取速度快,可提高怀山药多糖的提取效率。
(5)本发明采用乙醇/硫酸铵双水相法除蛋白,该体系不使用任何有机试剂,不存在有机试剂残留问题,且双水相含水量高(超过70%),一般不会引起生物活性物质失活或变性。
附图说明
图1为本发明怀山药多糖清DPPH活性的实验结果图( ±s,n=3).*p<0.05,**p<0.01vs Control。
图2为本发明怀山药多糖清除羟自由基活性的实验结果图(±s,n=3).*p<0.05,**p<0.01vs Control。
图3为本发明怀山药多糖抑制酪氨酸酶活性的实验结果图(±s,n=3).*p<0.05,**p<0.01vs Control。
图4为本发明怀山药多糖抑制人A-375黑色素细胞增殖的实验结果图(±s,n=3).*p<0.05,**p<0.01vs Control。
图5为本发明怀山药多糖抑制人A-375黑色素细胞黑色素合成的实验结果图(±s,n=3).*p<0.05,**p<0.01vs Control。
图6为本发明怀山药多对H2O2诱导损伤人永生化表皮细胞(HaCaT细胞)的保护作用的实验结果图(±s,n=3).*p<0.05,**p<0.01vs Control。
具体实施方式
下面结合图1至图3,对本发明做进一步说明:
实施例1:微波提取怀山药多糖
A、原料的处理:怀山药根茎部分洗净,在60℃烘箱烘干至恒重,粉碎机粉碎后过20目筛;
B、提取:山药粉和水按料液比1:25混合,80℃微波提取20分钟;将怀山药多糖提取液离心,所得滤渣再次重复提取1次,合并滤液,即为怀山药多糖水提液。
C、浓缩:将怀山药多糖水提液减压浓缩至原体积的五分之一。
D、醇沉:向怀山药多糖浓缩液中加入适量95%乙醇使其乙醇终浓度为80%,4℃放置过夜,可见白色沉淀析出,8000r/min离心5min,收集沉淀物。
F、除蛋白:将多糖沉淀物复溶于水,水溶液与乙醇、硫酸铵按照质量比5.159:2.811:2.03的比例配置成乙醇/硫酸铵双水相体系,倒入分液漏斗中,静止过夜,随后收集下相溶液。
G、透析:将下相溶液放入截留分子量为1000Da的透析袋中,先用自来水透析24h,随后在去离子水中透析24h,每4h换水一次。
H、冷冻干燥:将透析液减压浓缩后,冷冻干燥后即得怀山药多糖。按照苯酚-硫酸法测定多糖含量,每100g山药粉可得9.6g多糖;凝胶色谱排阻法测得分子量分布为10.3kDa-131.5 kDa。
试验例1:DPPH自由基清除实验
以抗坏血酸(VC)作为阳性对照,测定DPPH 自由基清除率。将样品或VC溶于去离子水中分别配制成6个梯度的怀山药多糖或VC溶液(10,100,1000,2000,3000,4000,5000μg/mL)。在试管中依次加入2mL上述溶液(空白管不加),2mL DPPH溶液(本底管不加),各管用蒸馏水补足至4mL,振荡器混匀,室温静置30min,在517nm处以蒸馏水调零测定吸光度(OD值),计算公式为:
式中:A DPPH 为未加入样品或VC的DPPH溶液的吸光度;A实验为DPPH溶液加入样品或VC后的吸光度;A本底为未加入DPPH的样品溶液的吸光度。
如图1所示,怀山药多糖具有清除DPPH活性的能力,DPPH清除活性随多糖浓度升高而升高。当样品浓度为5mg/mL时,DPPH清除率达到64.3%,表明怀山药多糖具有良好的抗氧化活性。
试验例2:羟自由基清除实验
以抗坏血酸(VC)作为阳性对照,测定羟自由基清除能力。将样品或VC溶于水中分别配制成6个梯度的怀山药多糖或VC溶液(10,100,1000,2000,3000,4000,5000μg/mL)。在试管中依次加入1mL上述溶液(空白管不加),1mL 2mmol/L的FeSO4溶液,1mL 6mmol/L的水杨酸-乙醇溶液,最后在各管加1mL 6mmol/L的H2O2启动反应(本底管不加),各管用蒸馏水补足至4mL,振荡器混匀,37℃水浴保温置30min,在510nm处以蒸馏水调零测定吸光度(OD值),计算公式如下:
式中:A空白为未加入样品或VC溶液的吸光度;A实验为加入样品或VC溶液的吸光度;A本底为未加入6mmol/L的H2O2启动反应的溶液的吸光度。
如图2所示,怀山药多糖具有清除羟自由基活性的能力,羟自由基清除活性随多糖浓度升高而升高。当样品浓度为5mg/mL时,羟自由基清除率达到58.4%,表明怀山药多糖具有良好的抗氧化活性。
试验例3:酪氨酸酶抑制实验
取对数生长期得人A-375黑色素细胞,用完全培养基调节细胞浓度为5×104个/mL,接种于96孔板,每孔100μL,24 h后给药组分别添加100 μL含有不同浓度怀山药多糖的培养基继续培养48h,同时设置添加空白完全培养基的对照组和含有不同浓度的熊果苷的阳性药物组,每孔设置3个复孔。培养到时间节点后,PBS洗涤2次,每孔加入10TritonX-100溶液90 μL.震荡5min溶解细胞,经37℃预温后每孔加入1%的L-Dopa10μL。37赋予30min。于490 nm波长下测吸光度值(OD值),以空白孔调零,计算酪氨酸酶活力。
如图3所示,怀山药多糖对酪氨酸买有抑制作用,且随着多糖浓度增加,酪氨酸酶活力下降,有明显的量效关系。怀山药多糖浓度在500μg/mL时酪氨酸酶活力为44.2%,与对照组相比具有极显著差异。表明怀山药多糖具有美白、淡斑等功效。
试验例4:人A-375黑色素细胞增殖抑制实验
取对数生长期得人A-375黑色素细胞,用完全培养基调节细胞浓度为5×104个/mL,接种于96孔板,每孔100μL,24 h后给药组分别添加100 μL含有不同浓度怀山药多糖的培养基继续培养48h,同时设置添加空白完全培养基的对照组和含有不同浓度的熊果苷的阳性药物组,每孔设置3个复孔。培养到时间节点后,每孔加入10μL CCK-8,37℃孵育20min,再450nm波长下测量各孔吸光度值,按照如下公式计算各孔黑色素细胞抑制率(%)。
黑色素细胞抑制率(%)=(1-给药组OD值/对照组OD值)×100%
如图4所示,怀山药多糖对黑色素细胞增殖具有抑制作用,怀山药多糖浓度在500μg/mL时,黑色素细胞存活率为含量为73.1%,与对照组相比具有极显著差异,且随着浓度且随着多糖浓度增加,黑色素细胞存活率下降,有明显的量效关系。表明怀山药多糖具有美白、淡斑等功效。
试验例5:人A-375黑色素细胞黑色素合成抑制实验
取对数生长期的人A-375黑色素细胞,用完全培养基调节细胞浓度为5×104个/mL,接种于96孔板,每孔100μL,24 h后给药组分别添加100 μL含有不同浓度怀山药多糖的培养基继续培养48h,同时设置添加空白完全培养基的对照组和含有不同浓度的熊果苷的阳性药物组,每孔设置6个复孔。培养到时间节点后,离弃上清,PBS洗涤,每组取3个复孔进行细胞计数,剩余3个复孔培养分别加入1mol/L NaOH溶液150μL于85℃水浴保温裂解1h,在490 nm波长下测量各孔吸光度值(OD值),按照如下公式计算各孔黑色素细胞中黑色素合成抑制率(%)。
黑色素合成抑制率(%)=[1-(给药组OD值/细胞数量)/(对照组OD值/细胞数量)]×100%
如图5所示,怀山药多糖对黑色素合成具有抑制作用,怀山药多糖浓度在500 μg/mL时,黑色素含量为60.6%,与对照组相比具有极显著差异,且随着多糖浓度增加,黑色素含量下降,有明显的量效关系。表明怀山药多糖具有美白、淡斑等功效。
试验例6:H2O2诱导损伤HaCaT细胞的保护实验
HaCaT细胞用完全培养基培养,取对数生长期细胞,按5×104个/mL接种于96孔板,每孔100μL,待细胞完全贴壁后,更换含250μM过氧化氢的DMED培养基继续培养4h,弃上清,PBS洗三遍,加入含50μg/mL、250μg/mL、500μg/mL怀山药多糖的DMED完全培养基继续培养24h,以4μg/mL维生素c为阳性对照。培养到时间节点后,每孔加入10μL CCK-8,37℃孵育20min,再450nm波长下测量各孔吸光度值,按照如下公式计算各孔存活率(%)。
HaCaT细胞存活率(%)=给药组OD值/对照组OD值×100%
如图6所示,怀山药多糖对H2O2诱导氧化损伤的HaCaT细胞有较好的保护作用,怀山药多糖浓度在500μg/mL时,HaCaT细胞活力88.3%,与对照组相比具有显著差异,且随着浓度且随着多糖浓度增加,HaCaT细胞活力增加,有明显的量效关系。表明怀山药多糖具有抵抗皮肤氧化损伤及老化功效。
综上所述,本发明所得怀山药多糖具有抗氧化、抗衰老、祛斑美白的功效,可用作化妆品和/或护肤品的添加剂,也可用于功能性食品添加剂。
上面结合附图对本发明进行了示例性的描述,显然本发明的实现并不受上述方式的限制,只要采用了本发明的方法构思和技术方案进行的各种改进,或未经改进将本发明的构思和技术方案直接应用于其它场合的,均在本发明的保护范围内。
Claims (5)
1.一种怀山药多糖的制备方法,包括以下步骤:
步骤1、怀山药烘干粉碎;
步骤2、微波提取怀山药多糖水提液;
步骤3、怀山药多糖水提取液浓缩;
步骤4、浓缩液的醇沉并收集沉淀物;
步骤5、沉淀物除蛋白,并收集下相溶液;
步骤6、透析;
步骤7、冷冻干燥即得怀山药多糖;
步骤1中怀山药根茎部分洗净,在60℃烘箱烘干至恒重,粉碎机粉碎后过20目筛;
步骤2中筛选后山药粉末和水按料液比1:25混合,80℃微波提取20分钟;将怀山药多糖提取液离心,所得滤渣再次重复提取1次,合并滤液,即为怀山药多糖水提取液;
步骤3中将怀山药多糖水提取液减压浓缩至原体积的五分之一;
步骤4中向怀山药多糖浓缩液中加入适量95%乙醇使其乙醇终浓度为80%,4℃放置过夜,可见白色沉淀析出,8000r/min离心5min,收集多糖沉淀物;
步骤5中将多糖沉淀物复溶于水,水溶液与乙醇、硫酸铵按照质量比5.159:2.811:2.03的比例配置成乙醇/硫酸铵双水相体系,倒入分液漏斗中,静止过夜,随后收集下相溶液;
步骤6中将下相溶液放入截留分子量为1000Da的透析袋中,先用自来水透析24h,随后在去离子水中透析24h,每4h换水一次;
步骤7中将透析液减压浓缩后,冷冻干燥后即得怀山药多糖;按照苯酚-硫酸法测定多糖含量,每100g山药粉可得9.6g多糖;凝胶色谱排阻法测得分子量分布为5kDa -500kDa。
2.一种怀山药多糖,其特征在于:使用权利要求1所述制备方法制得。
3.权利要求2所述怀山药多糖在制备具有美白功效的药品或保健品肤外用剂的应用。
4.权利要求2所述怀山药多糖在制备具有淡斑功效的药品、保健品或皮肤外用剂的应用。
5.权利要求2所述怀山药多糖在制备具有抗衰老功效的药品、保健品或皮肤外用剂的应用。
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