CN116807956A - 一种抗皱修护眼霜及其原料配方和应用 - Google Patents
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Abstract
本发明属于化妆品领域，具体涉及一种抗皱修护眼霜及其原料配方和应用。一种抗皱修护眼霜，所述眼霜的原料配方中含有沙棘干细胞提取物。本申请采用干细胞组织分离培养技术得到了沙棘形成层干细胞，进一步通过大量实验得到优化后的继代培养基，通过额外添加生物诱导子，在一定程度上促进了沙棘干细胞的活性次级代谢产物的形成。另外，通过实验检测发现，本申请所制备的沙棘干细胞提取物中活性成分含量高，且具有很强的抗氧化活性，能够抑制并淡化皱纹，与其他辅料按一定配方混合制备得到的眼霜吸收效果好，修复作用明显。
Description
技术领域
本发明属于化妆品领域，具体涉及一种抗皱修护眼霜及其原料配方和应用。
背景技术
植物是许多重要药用化学物质的来源。许多植物含有抗病毒、抗菌、抗癌和抗氧化等生理活性的化合物，对人类的肿瘤、衰老、心血管等多种疾病的防治具有重要意义。植物是探究新药的理想来源，因此越来越多的研究者关注植物次生代谢产物的生产。沙棘为胡颓子科落叶灌木或小乔木，分布在我国华北、西北、东北及西南等地，具有良好的经济价值和生态效益。沙棘含有多种维生素、黄酮类化合物、三萜及甾体类化合物、蛋白质、氨基酸、脂肪酸类、有机酸、糖和微量元素等多种物质。沙棘具有保护心血管、调节免疫力、保护肝脏、降血脂、抗炎、抗氧化等作用。
获取药用植物次生代谢产物的方法有许多种，目前多数是从药用植物中直接提取。但是很多药用植物生长缓慢，次生代谢产物的量低而且许多仅存于特定的植物器官中，难以适应现代制药工业生产的需求；采用化学合成也难以实现原料供应，因为很多次生代谢产物化学结构非常复杂和独特，涉及的生物合成途径繁杂，合成成本高。因此，尤其是对于一些珍稀濒危的中药来说，植物细胞培养依旧是获取次生代谢产物相对可行的选择。根据文献得知，利用诱导子来提高次生代谢产物，也是目前在药用植物细胞培养中常用的方法，其中最常用的是生物诱导子和非生物诱导子。生物诱导子主要包括病原菌(真菌、细菌、病毒与酵母)与植物细胞成分，其中主要是细胞壁水解产物，而非生物诱导子主要是起诱导作用的理化因子。
目前通过植物细胞培养成功的例子已经有很多，如从红豆杉中培养获得紫杉醇，从紫草中培养获得紫草素，从人参中培养获得人参皂苷等。尽管如此，植物细胞培养在工业生产中还存在很多局限性，如传统植物细胞培养需经历一个脱分化过程，但脱分化细胞的次生代谢产物丰度非常低，甚至根本没有，而且在这个过程中会发生体细胞突变，导致细胞系遗传稳定性降低；另外在长期培养过程中，细胞会受到环境的影响，增长慢、收益率低，阻碍其商业化生产。
植物干细胞又称为分生组织，是相对于植物体内已经分化的成熟组织而言的，是植物体内未分化的细胞。植物分生组织根据其位置可以分为顶端分生组织和侧生分生组织两大类。其中顶端分生组织包括茎尖分生组织和根尖分生组织；侧生分生组织包括维管形成层和木栓形成层。植物干细胞是生长发育的源泉和信号调控中心。它是未分化的细胞，具有很强的自我更新和持续分裂能力，是拥有永久生存能力的“不朽细胞”。植物干细胞培养技术是利用干细胞生长和遗传特性方面的优势，在传统细胞培养的基础上，以植物干细胞为目标，诱导、分离和培养外植体，建立相应的干细胞体系。
传统药用植物细胞培养需要经过脱分化过程，脱分化过程往往不彻底，出现部分分化现象；细胞不稳定，易出现体细胞突变现象，长期培养时，在培养初始阶段生长良好，但随着培养时间的延长，细胞生长速度降低，很多细胞出现褐变及死亡，细胞形态也发生变化；而药用植物干细胞培养的干细胞系在长期培养时，能保持稳定快速生长，形态也不会发生很大变化。当植物细胞以细胞团的形式而不是作为单个细胞被培养时，这种细胞团由于内外环境的差异，导致细胞生长发生变化。因此，在建立细胞的悬浮培养时，细胞的聚集度越低，越有利于细胞的生长。在进行大规模培养时，细胞容易受生物反应器中剪切力和细胞聚集的影响出现生长速度降低等问题。来自形成层的干细胞系含有大量的液泡，对剪切力灵敏度低，并且具有较低的聚集性，细胞生长能力不会受到影响。在低于-70℃的超低温条件下，有机体内部的生化反应极其缓慢，甚至终止。因此，采取适当的方法将生物材料降至超低温，即可使生命活动固定在某一阶段不衰老死亡。当以适当的方法将冻存的生物材料恢复至常温时，其内部的生化反应可恢复正常。传统组织培养的细胞在低温储藏之后存活率低，且恢复生长能力的延迟期漫长，而来自形成层的干细胞系在低温储藏后存活率很高，并能较快地重新生长。
药用植物干细胞培养作为一种新技术，是植物细胞培养的一个新阶段，其为解决传统细胞培养遇到的问题提供了一个全新的解决方案。但是到目前为止，药用植物干细胞培养仍然存在着一些不足和疑问。如来源于人参形成层的干细胞系提取物和培养物的抗衰老和抗氧化活性不是依赖于人参皂苷的作用，而且植物干细胞培养得到的细胞系中含有与常规野山参细胞不同的活性成分。但是由形成层得到的干细胞系是否含有与常规野山参不同的活性成分，以及为什么会合成新的活性物质，这种新的物质是什么，这一系列问题目前尚未得到更为深入的研究。
虽然已经利用植物干细胞培养技术成功得到某些药用植物的干细胞，但是植物不同，干细胞培养的建立方法也可能有所不同，因此还需要一定的实验对目前的方法进行完善。目前，药用植物干细胞技术由于各种原因还没有广泛应用。但是随着该技术的完善和推广，其巨大优势会逐渐显现出来，并将大大推动与之相关的食品、医药和化妆品等行业的发展。
发明内容
为了解决以上的技术问题，本申请提供了一种新的植物干细胞及其制备方法，并将其应用于抗皱修护的化妆品制备中，为化妆品领域提供了更多原料的选择，也进一步拓宽了干细胞植物的来源和应用。本申请采用干细胞组织分离培养技术得到了沙棘形成层干细胞，进一步通过大量实验得到优化后的继代培养基，通过额外添加生物诱导子，在一定程度上促进了沙棘干细胞的活性次级代谢产物的形成。另外，通过实验检测发现，本申请所制备的沙棘干细胞提取物中活性成分含量高，且具有很强的抗氧化活性，能够抑制并淡化皱纹，与其他辅料按一定配方混合制备得到的眼霜吸收效果好，修复作用明显。
为了实现上述的发明目的，本发明采用了以下的技术方案：
一种抗皱修护眼霜，所述眼霜的原料配方中含有沙棘干细胞提取物。
作为优选，所述眼霜的原料配方中还包含以下组分：甘油、透明质酸钠、1，2-己二醇、聚二甲基硅氧烷、鲸蜡硬脂醇、黄原胶、肉豆蔻酸和山梨酸钾。
作为优选，按重量份数计，所述眼霜包含以下组分：沙棘干细胞提取物10-20份，甘油5-8份，透明质酸钠2-5份，1，2-己二醇0.5-2.0份、聚二甲基硅氧烷0.5-1.0份、鲸蜡硬脂醇0.5-1.0份、黄原胶0.2-0.5份、肉豆蔻酸0.1-0.3份和山梨酸钾0.01-0.03份。
作为优选，所述沙棘干细胞提取物的制备方法包括以下步骤：
1)外植体消毒：选取沙棘外植体中生长状态良好的枝条，用洗洁精浸泡搓洗，再用蒸馏水搓洗干净，滤纸吸干外植体表面水分，将嫩茎剪成1cm小段，用75％酒精浸泡，无菌水清洗3次，然后用2％的HgCl浸泡，无菌水清洗5次备用；
2)无菌接种和培养：在超净工作台中将步骤1)消毒好的茎段表皮撕下，切成块状，倒置放于WPM培养基中，在培养箱中25℃，100r/min震荡暗箱培养，14天后将形成层干细胞转移至继代培养基中，每21天继代一次，得到沙棘形成层干细胞；
3)收集培养沙棘形成层干细胞后的继代培养基，过滤浓缩后保留其中的活性成分，将该培养基浓缩液和沙棘形成层干细胞的裂解物保存于4℃备用，即为沙棘干细胞提取物。
作为优选，所述继代培养基的制备方法为：以WPM培养基为基础培养基，进一步添加生物诱导子。
作为优选，所述WPM培养基的配方为：20g/L蔗糖、1.8mg/LNAA、0.1g/LVc、300mg/LL-谷氨酰胺和8g/L琼脂，pH为5.8。
作为优选，所述生物诱导子为尖孢镰刀菌和黑曲霉中的一种或两种。
作为优选，所述生物诱导子为尖孢镰刀菌。
作为优选，所述尖孢镰刀菌的用量为20-40mg/L。
进一步，本申请提供了所述的沙棘干细胞提取物在制备抗皱修复化妆品中的应用。
采用本技术方案的有益效果是：首先，本申请主要是针对沙棘干细胞设计的培养基和培养方法，旨在得到活性更强，含量更高的沙棘干细胞提取物，其中通过多种生物诱导子的筛选得到了两种菌株，尤其是添加一定浓度的尖孢镰刀菌后，能够极大地促进沙棘干细胞继代培养过程中活性物质的生成。通过本申请制备得到的沙棘干细胞提取物经试验检测，证明其具有很强的自由基清除率，能够减少皱纹产生，同时促进成纤维细胞的增殖。其次，由于沙棘干细胞中的活性物质大部分为酚类，在应用过程中易产生变质问题，但通过本申请的配方制备得到的眼霜稳定性较好，具有很好的市场发展前景。
具体实施方式
下面将结合本发明实施例，对本发明实施例中的技术方案进行清查、完整的描述，进而进一步解释发明。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部实施例。给予本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1沙棘干细胞提取物制备
原料：沙棘购自呼伦贝尔产地，黑曲霉和尖孢镰刀菌来自中国普通微生物菌种保藏管理中心，蔗糖、NAA、Vc、L-谷氨酰胺、琼脂、甘油、透明质酸钠、1，2-己二醇、聚二甲基硅氧烷、鲸蜡硬脂醇、黄原胶、肉豆蔻酸和山梨酸钾购自市售产品。
制备方法：
1)外植体消毒：选取沙棘外植体中生长状态良好的枝条，用洗洁精浸泡搓洗，再用蒸馏水搓洗干净，滤纸吸干外植体表面水分，将嫩茎剪成1cm小段，用75％酒精浸泡，无菌水清洗3次，然后用2％的HgCl浸泡，无菌水清洗5次备用；
2)无菌接种和培养：在超净工作台中将步骤1)消毒好的茎段表皮撕下，切成块状，倒置放于WPM培养基中，在培养箱中25℃，100r/min震荡暗箱培养，14天后将形成层干细胞转移至继代培养基中，每21天继代一次，得到沙棘形成层干细胞；
3)收集培养沙棘形成层干细胞后的继代培养基，过滤浓缩后保留其中的活性成分，将该培养基浓缩液和沙棘形成层干细胞的裂解物保存于4℃备用，即为沙棘干细胞提取物。
其中，WPM培养基的配方为：20g/L蔗糖、1.8mg/LNAA、0.1g/LVc、300mg/LL-谷氨酰胺和8g/L琼脂，pH为5.8。
继代培养基的制备方法为：以WPM培养基为基础培养基，进一步添加生物诱导子。
本申请实施例仅展示采用以下几种不同的生物诱导子培养基培养得到的沙棘干细胞提取物，均采用上述制备方法得到，除使用的生物诱导子及其浓度不同，其余步骤均一致。
沙棘干细胞提取物1：尖孢镰刀菌40mg/L；
沙棘干细胞提取物2：尖孢镰刀菌60mg/L
沙棘干细胞提取物3：黑曲霉60mg/L；
沙棘干细胞提取物4：尖孢镰刀菌20mg/L和黑曲霉60mg/L；
沙棘干细胞提取物5：酵母提取物10mg/L；
沙棘干细胞提取物6：花生四烯酸1mg/L；
沙棘干细胞提取物7：无生物诱导子添加。
实施例2活性成分含量测定
糖测定：参照GB5009.8—2016《食品安全国家标准食品中果糖、葡萄糖、蔗糖、麦芽糖、乳糖的测定》的方法，采用示差折光检测器，ZXBridgeAmide色谱柱(3.5μm，4.6mm×250mm)，柱温保持40℃，进样量为10μL；流动相为90％乙腈水∶0.1％氨水，流速为1.0mL/min。分别取果糖、葡萄糖、蔗糖、鼠李糖和山梨醇用水配制成质量浓度20mg/mL的标准溶液，稀释至不同浓度进样，根据浓度和相应峰面积得到标准曲线。准确移取1mL沙棘干细胞提取物至50mL离心管，加10mL水后，再依次加入2mL硫酸锌和2mL亚铁氰化钾，涡旋1min，用水定容至25mL，10000r/min离心5min后取上清液，0.45μm微孔滤膜过滤后直接进样。
有机酸测定：高效液相色谱条件：采用紫外检测器，InertSustain-C18色谱柱(4.6mm×250mm，5μm)，进样量为20μL，柱温保持在30℃；流动相为0.05mol/LKH2PO4溶液(用20％磷酸调为pH2.70)，等度洗脱20min，流速为0.6mL/min。取草酸、抗坏血酸、乳酸、奎宁酸、苹果酸和柠檬酸标准溶液用流动相稀释至不同浓度的后分别进样，根据浓度和相应峰面积得到有机酸标准曲线。将沙棘干细胞提取物用流动相稀释10倍，然后用0.45μm微孔滤膜过滤后进样。
总黄酮测定：参照DB43T476—2009《植物源性食品中总黄酮的测定》。取芦丁用水配制成不同浓度的标准溶液，各取1mL依次加入2.0mL三氯化铝溶液(2.5g/100mL)，2.0mL醋酸钾溶液(9.82g/100mL)，混匀，体积分数30％的乙醇定容至50mL，静置15min，以水为空白，于波长415nm处测吸光度值，绘制芦丁标准曲线。沙棘干细胞提取物(4000r/min)离心10min，移取1mL上清液置于50mL容量瓶中，同上操作，于波长415nm处测吸光度值。总黄酮含量以芦丁计。
总酚测定：采用Folin-ciocalteu法，将没食子酸标准溶液稀释至不同浓度，各取0.5mL上述浓度的标液，加2.5mL蒸馏水，再加入0.5mLFolin-ciocalteu显色剂和1.5mL7.5％Na2CO3溶液，混合均匀。室温避光1h后，于波长765nm处测定吸光度值，以蒸馏水作为空白对照，绘制没食子酸标准曲线。将沙棘干细胞提取物4000r/min离心10min，准确移取0.5mL上清液，按照上述方法进行显色反应，于波长765nm处测定吸光度值。总酚含量以没食子酸计。
表1不同样品中活性成分的含量
实施例3抗氧化活性检测
取沙棘干细胞提取物样品分别配制浓度为1.0mg/mL溶液进行体外抗氧化测定；
DPPH自由基(DPPH·)清除能力测定：参照Nagai等方法，分别量取2mL不同样品溶液于具塞试管中，加入浓度为0.2mmol/LDPPH甲醇溶液1mL。密封后混匀，于25℃恒温并避光反应1h，测定517nm处的吸光值Ai。用等体积的甲醇溶液代替DPPH溶液为样品对照组Aj，消除样品溶液对吸光值的影响，等体积的蒸馏水代替样品溶液作空白对照，测吸光度为A0，清除率(％)＝1-(Ai-Aj)/A0×100。
结果如表2所示，本申请制备的沙棘干细胞提取物的自由基清除率达到了96％以上，具有较高的抗氧化活性。其中选用的不同生物诱导子所得到的沙棘干细胞提取物样品的活性有较大差别，本申请实验中，酵母提取物和花生四烯酸对沙棘干细胞的诱导作用较弱，黑曲霉和尖孢镰刀菌分别单独使用的诱导作用较强，但共同诱导时并无明显协同作用，活性还略有下降。另外，当添加的尖孢镰刀菌的浓度为50mg/L时，可以看到自由基清除率下降至82.07％，结合表1实验数据发现，其中沙棘干细胞提取物2样品中黄酮类和酚类成分含量也有较大程度的下降，说明生物诱导子的浓度也是影响沙棘干细胞活性成分生成的一个重要因素，且同时本申请的诱导子属于最适浓度型，即活性物质的合成要求诱导子处于一个合适的浓度下，才能表现出较强的活性。
表2不同样品的抗氧化能力测定
实施例4眼霜的制备及其抗皱修复活性测定
眼霜制备：将透明质酸钠、甘油、1，2-己二醇和去离子水搅拌混合均匀后加热至85℃，作为A相；将鲸蜡硬脂醇和肉豆蔻酸混合均匀加热至85℃，作为B相；将B相加入到A相，乳化的同时加入聚二甲基硅氧烷、黄原胶搅拌30min，控制搅拌速率为1200r/min，降温至45℃时加入山梨酸钾，搅拌均匀后，调节pH为6.0-7.0，降至室温后出料。
采用化妆品功效评价基本方法，对眼霜样品进行测试。
选择年龄35-55岁之间眼部有细小皱纹的女性30名进行测试。具体测试步骤如下：
(1)受试者在测试者指导下使用洗面奶清洁全脸，用清水将泡沫冲洗干净后用纸巾或干毛巾吸干多余水分。
(2)将受试者的双眼眼尾定为A点，双眼眼下定为B点。
(3)受试者在恒温恒湿房间中等待15分钟后开始测试。
(4)用皮肤测试仪记录受试者眼周皮肤各项指标。
(5)受试者在测试当天即开始使用眼霜，要求早晚各使用一次，连续使用3个月至测试结束。
表3眼霜使用前后眼周皮肤各指标的变化情况
从表3的实验数据可以发现，使用本申请制备的眼霜后，受试者眼周肌肤中关于水分、弹性和胶原蛋白的指标均有提升，且黑色素含量和皱纹评分大幅下降，证明了该眼霜具有很好的抗皱活性，对眼周受损肌肤具有较好的修复功能。
Claims (10)
1.一种抗皱修护眼霜，其特征在于，所述眼霜的原料配方中含有沙棘干细胞提取物。
2.根据权利要求1所述的眼霜，其特征在于，所述眼霜的原料配方中还包含以下组分：甘油、透明质酸钠、1，2-己二醇、聚二甲基硅氧烷、鲸蜡硬脂醇、黄原胶、肉豆蔻酸和山梨酸钾。
3.根据权利要求1或2所述的眼霜，其特征在于，按重量份数计，所述眼霜包含以下组分：沙棘干细胞提取物10-20份，甘油5-8份，透明质酸钠2-5份，1，2-己二醇0.5-2.0份、聚二甲基硅氧烷0.5-1.0份、鲸蜡硬脂醇0.5-1.0份、黄原胶0.2-0.5份、肉豆蔻酸0.1-0.3份和山梨酸钾0.01-0.03份。
4.根据权利要求3所述的眼霜，其特征在于，所述沙棘干细胞提取物的制备方法包括以下步骤：
1)外植体消毒：选取沙棘外植体中生长状态良好的枝条，用洗洁精浸泡搓洗，再用蒸馏水搓洗干净，滤纸吸干外植体表面水分，将嫩茎剪成1cm小段，用75％酒精浸泡，无菌水清洗3次，然后用2％的HgCl浸泡，无菌水清洗5次备用；
2)无菌接种和培养：在超净工作台中将步骤1)消毒好的茎段表皮撕下，切成块状，倒置放于WPM培养基中，在培养箱中25℃，100r/min震荡暗箱培养，14天后将形成层干细胞转移至继代培养基中，每21天继代一次，得到沙棘形成层干细胞；
3)收集培养沙棘形成层干细胞后的继代培养基，过滤浓缩后保留其中的活性成分，将该培养基浓缩液和沙棘形成层干细胞的裂解物保存于4℃备用，即为沙棘干细胞提取物。
5.根据权利要求4所述的眼霜，其特征在于，所述继代培养基的制备方法为：以WPM培养基为基础培养基，进一步添加生物诱导子。
6.根据权利要求5所述的眼霜，其特征在于，所述WPM培养基的配方为：20g/L蔗糖、1.8mg/L NAA、0.1g/L Vc、300mg/L L-谷氨酰胺和8g/L琼脂，pH为5.8。
7.根据权利要求5所述的眼霜，其特征在于，所述生物诱导子为尖孢镰刀菌和黑曲霉中的一种或两种。
8.根据权利要求7所述的眼霜，其特征在于，所述生物诱导子为尖孢镰刀菌。
9.根据权利要求8所述的眼霜，其特征在于，所述尖孢镰刀菌的用量为20-40mg/L。
10.权利要求4-9任意一项权利要求所述的沙棘干细胞提取物在制备抗皱修复化妆品中的应用。
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