CN114146015B - 一种含有花胶低聚肽的祛斑美容组合物及其制备方法和用途 - Google Patents
一种含有花胶低聚肽的祛斑美容组合物及其制备方法和用途 Download PDFInfo
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Abstract
一种含有花胶低聚肽的祛斑美容组合物及其制备方法和用途,属于食品领域,其组分包括花胶低聚肽粉、当归粉、桃仁粉、海参低聚肽粉、枸杞粉、茯苓粉、山药粉、酸枣仁粉、玫瑰花、葡萄籽提取物、维生素E。本发明在组方成分协同作用下,具有良好的祛斑美容作用。
Description
技术领域
本发明属于食品领域,具体涉及一种含有花胶低聚肽的祛斑美容组合物及其制备方法。
背景技术
人体最大的器官就是皮肤,皮肤颜色的差异主要取决于表皮黑素细胞合成的黑素情况。色斑通常是黑色素沉着导致的皮肤问题或疾病,常见的如黄褐斑、雀斑,是一类全球性问题。调查显示仅美国的黄褐斑人群就超过500万,中国的患者群体也呈逐年递增的趋势。
皮肤色斑的形成与多种因素有关,如遗传因素、皮肤抗氧化系统失衡、内分泌紊乱(甲状腺激素、性激素异常)、血管因素、月经不调、口服避孕药、妊娠、营养素异常、光损伤、精神心理状态等,具体表现出黑素细胞增多,分泌的黑素颗粒增加,色素堆积沉着在局部组织中。研究发现黑色素的分布到皮肤需要以下过程:首先黑色素小体形成、成熟,然后黑色素合成,黑色素细胞将含有大量黑色素的黑色素小体转运到邻近的皮肤角质细胞,角质细胞发生一些生理生化变化,导致黑色素在皮肤角质细胞内沉积。
目前祛斑的方法主要分三类:口服、外用、物理疗法,主要机理有:1、通过抑制或干扰黑色素的形成,达到防止色斑形成的目的;2、防晒,减少紫外线损伤;3、通过激光祛除皮肤中存在的黑素颗粒。
口服产品主要分两类,一是各种纯中药组合物,如CN201110423325.7一种祛斑养颜的中药组合物及其制备方法、CN201310751085.2一种美白祛斑的中药组合物及其制备方法;另一种是添加谷胱甘肽、维生素类等成分的组合物,如CN201710763074.4一种美白祛斑组合物及其应用,CN201010292567.2一种用于美白祛斑的组合物及其制备方法。因作用单一,效果不明显。
外用的主要是各种化妆品、药膏等,如CN201510450427.6一种美白祛斑复方制剂及其制备方法,CN201310527260.X一种美白祛斑乳液,通常添加壬二酸、熊果苷、对甲氧酚、氨甲环酸等成分或防晒成分,只能防止色斑加重或减轻颜色,治疗效果差,而且但是壬二酸、对甲氧酚大多数有一定副作用。
物理疗法一般是激光治疗、光子嫩肤,并且必须严格防晒,通常使用防晒霜来配合使用,存在疗效不稳定、引发炎症等问题。
虽然色斑对身体健康没有影响,但因其多出现在面部、颈部,十分影响容貌形象,甚至引发严重的社交、心理、精神等问题。爱美是人的天性,祛斑是有斑人士永恒的追求,因此开发一款安全高效祛斑美容产品具有广阔的市场前景。
花胶,也称鱼胶,与燕窝、鱼翅齐名,是“八珍”之一,《本草纲目》记载:花胶能补肾益精,滋养筋脉,能治疗肾虚滑精及产后风痉。花胶主要成分为高级胶原蛋白,超过80%,还含有多种维生素及钙、锌、铁、硒等微量元素,自古就是美容养颜的佳品,具有滋阴养颜,补血补肾,强壮机能之效。花胶蛋白通过不同的蛋白酶酶解可获得多种活性肽段,目前已有花胶肽的制备和应用研究,如CN201710907667.3一种花胶多肽制备方法,CN201810406679.2一种产后调理的花胶弹性蛋白肽组合物及其制备方法,但是对花胶肽具体活性肽段的研究及祛斑美容组方的应用未见报道。
发明内容
本发明针对上述存在问题、现状及发展前景,提供一种含有花胶低聚肽的祛斑美容组合物,具有祛斑美容的效果。
本发明的技术方案如下:一种含有花胶低聚肽的祛斑美容组合物,包括花胶低聚肽粉50-100份、当归粉20-40份、桃仁粉30-50份、海参低聚肽粉10-30份、枸杞粉30-50份、茯苓粉20-40份、山药粉20-40份、酸枣仁粉20-50份、玫瑰粉20-30份。
进一步的,所述的花胶花胶低聚肽粉为花胶小分子活性肽粉。
进一步的,花胶低聚肽基于如下方式制备而得:花胶处理后,加入15~20倍质量体积的水制成匀浆液置于酶解罐中,然后加入花胶蛋白质质量3~5%的复合蛋白酶,在50~55℃下酶解3~5小时,酶反应pH值控制在7.0~8.0,酶解结束后升温至80~90℃灭酶10分钟,得到花胶蛋白酶解液,将蛋白酶解液8000转/分钟离心10分钟,去除颗粒状物质,然后采用膜分离技术进行分离,截留分子量为3000Da,过膜液喷雾干燥得花胶低聚肽粉:所述复合蛋白酶的质量配比为:中性蛋白酶:胰蛋白酶:菠萝蛋白酶:风味蛋白酶=(4~6):(3~5):(1~3):(2~4)。
进一步的,花胶低聚肽中90%以上肽的分子量小于1000Da。
进一步的,花胶小分子活性肽由花胶低聚肽分离纯化而得:将花胶低聚肽粉加水溶解,配制成浓度为100mg/mL的溶液,采用葡聚糖凝胶Sephadex LH-20柱色谱法进行分离纯化,流动相为40%甲醇,流速为0.4~0.6mL/min,洗脱液280nm测定吸光度,根据吸光度值收集所需峰;采用高效液相色谱进一步纯化,色谱条件如下:C18色谱柱,流动相A为含体积百分数0.06%~0.08%三氟乙酸水,流动相B为乙腈,梯度洗脱条件为:0~15min,3%B;15~20min,3%~10%B;20~30min,10%B~20%B;30~40min,20%B~35%B;流速为0.8mL/min,检测波长为280nm,收集保留时间在26分钟的色谱峰,浓缩后冷冻干燥得花胶小分子活性肽。
进一步的,花胶小分子活性肽氨基酸序列为:Gly-Pro-Leu-Ala-Gly-Pro。
进一步的,海参低聚肽基于如下方式制备而得:海参处理后,加入15~20倍质量体积的水制成匀浆液置于酶解罐中,然后加入海参质量2~5%的复合蛋白酶,在40~50℃下酶解4小时,酶反应pH值控制在8.0~9.0,酶解结束后升温至80~90℃灭酶10分钟,得到海参蛋白酶解液,将蛋白酶解液8000转/分钟离心10分钟,去除颗粒状物质,然后采用膜分离技术进行分离,截留分子量为3000Da,过膜液喷雾干燥得海参低聚肽粉;所述复合蛋白酶的质量配比为:中性蛋白酶:木瓜蛋白酶:风味蛋白酶=(2~4):(3~5):(3~5)。
进一步的,海参低聚肽中90%以上肽的分子量小于1000Da。
进一步的,美容组合物为粉剂、颗粒剂、片剂、胶囊剂、丸剂、口服溶液中的一种。
一种所述的美容组合物在祛斑美容食物、化妆品或药物中的应用。
酪氨酸酶(TYR)在黑色素合成过程中其到关键作用,是重要的限速酶。当酪氨酸酶过表达时就会导致黑色素增多、异常,在皮肤沉着皮肤色斑。合成黑色素的开始源于L-酪氨酸的氧化,TYR可将L-酪氨酸氧化为多巴醌,多巴醌通过自氧化转变为多巴色素,多巴色素的产物(5,6-二羟基吲哚羧酸与5,6-二羟基吲哚)进一步被氧化成真黑素。通常不同肤色的黑色素细胞中酪氨酸酶表达量基本无差异,因此想要达到好的祛斑效果,对酪氨酸酶活性进行抑制是关键所在。
花胶低聚肽中的酪氨酸酶抑制活性肽(Gly-Pro-Leu-Ala-Gly-Pro)能够直接干预黑色素的合成过程,通过抑制酪氨酸酶的活性,降低L-多巴、多巴醌以及5,6-吲哚醌等的产生,最终减少黑色素的形成,去除色斑。皮肤的光损伤也是色斑的影响因素。花胶低聚肽还能够通过清除自由基、提高抗氧化酶的活力、降低脂质过氧化物含量,起到抗氧化,延缓皮肤衰老的作用。同时花胶低聚肽粉本身属于胶原蛋白肽,能够为皮肤提供充足的营养物质。
中医认为色斑的产生与气血、脏器有关,是气滞血瘀,肝、脾、肾功能失调所致。现代研究表明血管因素也是色斑产生的重要因素之一。因此从活血,调理肝肾脾入手,对祛斑美容有良好的辅助作用。
当归粉是由当归制成的粉。当归性温,味甘、辛,归肝、心、脾经,有补血活血,润肠通便之功效,当归中的丁基苯酞能改善血液循环,当归多糖可直接和(或)间接促进机体造血。
桃仁粉是由桃仁制成的粉。桃仁性平,味苦、甘,归心、肝、大肠经,能够活血祛瘀,润肠通便。桃仁通过改善血流动力学(如增加灌流液流量和血流量、减少血管阻力),起到活血化瘀的作用。
酸枣仁粉是由酸枣仁制成的粉。酸枣仁性平,味甘、酸,归肝、胆、心经,具有养心补肝,宁心安神的功效。同时酸枣仁的皂苷、阿魏酸成分能够调节心血管、清除自由基。
玫瑰粉是由玫瑰花制成的粉。玫瑰花性温,味甘、微苦,归肝、脾经,具有行气解郁,疏肝和血的功效。
海参性温,味咸,入肾经,具有生百脉血,补肾益精,通肠润燥的功效。海参低聚肽粉是海参通过生物酶解技术制备的小分子活性肽,同时保留了海参皂苷、海参多糖等成分,营养丰富,能被人体完全吸收,起到滋补营养,养血补肾的作用。同时研究发现,海参低聚肽粉还具有降低B16黑素瘤细胞黑素含量、细胞修复、抗衰老的作用。
枸杞粉是由枸杞制成的粉。枸杞性平,味甘,归肝、肾经,能够滋补肝肾,益精明目。
茯苓粉是由茯苓制成的粉。茯苓性平,味甘、淡,归心、肺、脾、肾经,能够利水渗湿,健脾宁心。茯苓中的三萜类成分对脾虚治疗效果非常好。
山药粉是由山药制成的粉。山药性平,味甘,归脾、肺、肾经,有补脾养胃,补肾益肺的作用。山药中还含有多种氨基酸、多糖、微量元素等有益营养物质。
本发明的有益效果:
(1)本发明提供了一种新的花胶来源的酪氨酸酶抑制活性肽的制备、分离纯化工艺。该活性肽的分子量小,活性高,分离纯化步骤简单,易于获得,属食源性的活性肽,长期食用可达到祛斑美白的目的,有广阔的应用前景。
(2)本发明在前期对花胶低聚肽进行高效液相色谱-质谱分析鉴定和利用在线数据库进行活性筛选,获得高活性小分子肽,在此基础上利Sephadex LH-20凝胶柱层析及高效液相色谱等分离技术对此活性肽进行分离纯化,验证其活性,最终获得一个高酪氨酸酶抑制活性肽,其氨基酸序列为:Gly-Pro-Leu-Ala-Gly-Pro,经在线数据库BIOPEP及EROP-Moscow检索,所述序列为新的小分子活性肽。
(3)色斑产生的表现是黑色素增多沉积,是身体气滞血瘀,肝、脾、肾功能失调所致。本发明在直接降低黑色素含量的同时,改善血管因素,调理肝脾肾功能,间接减少色斑产生的条件,协同作用,达到祛斑美容的功效。花胶低聚肽中的酪氨酸酶抑制活性肽(Gly-Pro-Leu-Ala-Gly-Pro)通过抑制酪氨酸酶的活性,直接干预黑色素的合成过程,减少黑色素的形成。当归粉、桃仁粉、海参低聚肽粉、枸杞粉、茯苓粉、山药粉、酸枣仁粉、玫瑰粉,科学配伍,改善血管因素,活血、疏肝、补肾、健脾,使身体机能恢复平衡。
附图说明
图1是活性肽具有明显的酪氨酸酶抑制活性实验图。
具体实施方式
实施例1
一种含有花胶低聚肽的祛斑美容组合物中,含有酪氨酸酶抑制活性肽的花胶低聚肽的分离纯化方法,包括以下步骤:
(1)花胶处理后,加入20倍质量体积的水制成匀浆液置于酶解罐中,然后加入花胶蛋白质质量4%的复合蛋白酶,在50℃下酶解4小时,酶反应pH值控制在7.5,酶解结束后升温至90℃灭酶10分钟,得到花胶蛋白酶解液,将蛋白酶解液8000转/分钟离心10分钟,去除颗粒状物质,然后采用膜分离技术进行分离,截留分子量为3000Da,过膜液喷雾干燥得花胶低聚肽粉;所述复合蛋白酶的质量配比为:中性蛋白酶:胰蛋白酶:菠萝蛋白酶:风味蛋白酶=4:4:1:3。
(2)将(1)中的花胶低聚肽粉加水溶解,配制成浓度为100mg/mL的溶液,采用葡聚糖凝胶Sephadex LH-20柱色谱法(3.0×100cm)进行分离纯化,流动相为40%甲醇,流速为0.4mL/min,洗脱液280nm测定吸光度,根据吸光度值收集所需峰;采用高效液相色谱进一步纯化,色谱条件如下:依利特C18色谱柱(4.6mm×250mm,5μm),流动相A为0.08%三氟乙酸水(v/v),流动相B为乙腈,梯度洗脱条件为:0~15min,3%B,15~20min,3%~10%B,20~30min,10%B~20%B,30~40min,20%B~35%B,流速为0.8mL/min,检测波长为280nm,收集保留时间在26分钟的色谱峰,浓缩后冷冻干燥得花胶小分子活性肽。
步骤(1)得到的花胶低聚肽中分子量小于1000Da的肽占比93.8%。
步骤(2)得到的花胶小分子活性肽经液相色谱检测为单一峰,利用高效液相色谱-质谱测定结构,其氨基酸序列为:Gly-Pro-Leu-Ala-Gly-Pro。
花胶低聚肽的原料来源为黄花鱼(Larimichthys)、鳕鱼、赤嘴鳘、石斑鱼、黄姑鱼、鲟鱼、鲈鱼等其中的一种或几种。
酪氨酸酶抑制活性测定:
预先把冷冻干燥后的花胶小分子活性肽用蒸馏水配制成浓度为5mg/mL的溶液,依次稀释成浓度为1mg/mL、0.8mg/mL、0.6mg/mL、0.4mg/mL,0.2mg/mL样品溶液。底物L-酪氨酸(L-Tyr)用0.1mol/L磷酸盐缓冲溶液(pH=6.8)配制成0.5%浓度的L-Tyr溶液。酪氨酸酶用0.1mol/L磷酸盐缓冲溶液(pH=6.8)配制成100U/mL的溶液。
试验组:分别取不同浓度的样品溶液1mL,先加入0.5mLL-Tyr底物溶液,然后加入0.5mL酪氨酸酶溶液,37℃反应30min,在475nm处测定的吸光度A1。
试验空白对照组:分别取不同浓度的样品溶液1mL,先加入0.5mLL-Tyr底物溶液,然后加入0.5mL磷酸盐缓冲溶液(pH=6.8),37℃反应30min,在475nm处测定的吸光度A2。
阴性对照组:取1mL磷酸盐缓冲溶液(pH=6.8),先加入0.5mLL-Tyr底物溶液,然后加入0.5mL酪氨酸酶溶液,37℃反应30min,在475nm处测定的吸光度A3。
空白对照组:取1.5mL磷酸盐缓冲溶液(pH=6.8),加入0.5mLL-Tyr底物溶液,37℃反应30min,在475nm处测定的吸光度A4。
根据吸光度值计算不同浓度的小分子肽的酪氨酸酶抑制率。
抑制率=[(A3-A4)-(A1-A2)]/(A3-A4)×100%。
实验结果如图1所示,结果表明,该小分子活性肽具有明显的酪氨酸酶抑制活性,其IC50为0.318mg/mL。
实施例2
一种含有花胶低聚肽的祛斑美容组合物,组分为花胶低聚肽粉90份、当归粉30份、桃仁粉40份、海参低聚肽粉25份、枸杞粉50份、茯苓粉30份、山药粉40份、酸枣仁粉40份、玫瑰粉30份。
制备方法为:
(1)花胶低聚肽的制备方法:花胶处理后,加入18倍质量体积的水制成匀浆液置于酶解罐中,然后加入花胶蛋白质质量3%的复合蛋白酶,在52℃下酶解5小时,酶反应pH值控制在7.0,酶解结束后升温至85℃灭酶10分钟,得到花胶蛋白酶解液,将蛋白酶解液8000转/分钟离心10分钟,去除颗粒状物质,然后采用膜分离技术进行分离,截留分子量为3000Da,过膜液喷雾干燥得花胶低聚肽粉;所述复合蛋白酶的质量配比为:中性蛋白酶:胰蛋白酶:菠萝蛋白酶:风味蛋白酶=4:3:2:4。
(2)花胶低聚肽的分离纯化方法:将(1)中的花胶低聚肽粉加水溶解,配制成浓度为100mg/mL的溶液,采用葡聚糖凝胶Sephadex LH-20柱色谱法(3.0×100cm)进行分离纯化,流动相为40%甲醇,流速为0.5mL/min,洗脱液280nm测定吸光度,根据吸光度值收集所需峰;采用高效液相色谱进一步纯化,色谱条件如下:依利特C18色谱柱(4.6mm×250mm,5μm),流动相A为0.07%三氟乙酸水(v/v),流动相B为乙腈,梯度洗脱条件为:0~15min,3%B,15~20min,3%~10%B,20~30min,10%B~20%B,30~40min,20%B~35%B,流速为0.8mL/min,检测波长为280nm,收集保留时间在26分钟的色谱峰,浓缩后冷冻干燥得花胶小分子活性肽。
(3)海参低聚肽粉的制备方法:海参处理后,加入20倍质量体积的水制成匀浆液置于酶解罐中,然后加入海参质量4%的复合蛋白酶,在48℃下酶解4小时,酶反应pH值控制在8.0,酶解结束后升温至90℃灭酶10分钟,得到海参蛋白酶解液,将蛋白酶解液8000转/分钟离心10分钟,去除颗粒状物质,然后采用膜分离技术进行分离,截留分子量为3000Da,过膜液喷雾干燥得海参低聚肽粉;所述复合蛋白酶的质量配比为:中性蛋白酶:木瓜蛋白酶:风味蛋白酶=3:3:4。
(4)取花胶低聚肽粉90份、当归粉30份、桃仁粉40份、海参低聚肽粉25份、枸杞粉50份、茯苓粉30份、山药粉40份、酸枣仁粉40份、玫瑰粉30份,于三维运动混合机中混合15min,充分混匀,得到粉剂。
实施例3
一种含有花胶低聚肽的祛斑美容组合物,组分为花胶低聚肽粉70份、当归粉28份、桃仁粉35份、海参低聚肽粉20份、枸杞粉40份、茯苓粉25份、山药粉30份、酸枣仁粉35份、玫瑰粉20份。
制备方法为:
(1)花胶低聚肽粉的制备方法:花胶处理后,加入16倍质量体积的水制成匀浆液置于酶解罐中,然后加入花胶蛋白质质量5%的复合蛋白酶,在55℃下酶解3小时,酶反应pH值控制在8.0,酶解结束后升温至80℃灭酶10分钟,得到花胶蛋白酶解液,将蛋白酶解液8000转/分钟离心10分钟,去除颗粒状物质,然后采用膜分离技术进行分离,截留分子量为3000Da,过膜液喷雾干燥得花胶低聚肽粉;所述复合蛋白酶的质量配比为:中性蛋白酶:胰蛋白酶:菠萝蛋白酶:风味蛋白酶=6:3:3:3。
(2)花胶低聚肽的分离纯化方法:将(1)中的花胶低聚肽粉加水溶解,配制成浓度为100mg/mL的溶液,采用葡聚糖凝胶Sephadex LH-20柱色谱法(3.0×100cm)进行分离纯化,流动相为40%甲醇,流速为0.6mL/min,洗脱液280nm测定吸光度,根据吸光度值收集所需峰;采用高效液相色谱进一步纯化,色谱条件如下:依利特C18色谱柱(4.6mm×250mm,5μm),流动相A为0.07%三氟乙酸水(v/v),流动相B为乙腈,梯度洗脱条件为:0~15min,3%B,15~20min,3%~10%B,20~30min,10%B~20%B,30~40min,20%B~35%B,流速为0.8mL/min,检测波长为280nm,收集保留时间在26分钟的色谱峰,浓缩后冷冻干燥得花胶小分子活性肽。
(3)海参低聚肽粉的制备方法:海参处理后,加入15倍质量体积的水制成匀浆液置于酶解罐中,然后加入海参质量5%的复合蛋白酶,在50℃下酶解4小时,酶反应pH值控制在8.5,酶解结束后升温至85℃灭酶10分钟,得到海参蛋白酶解液,将蛋白酶解液8000转/分钟离心10分钟,去除颗粒状物质,然后采用膜分离技术进行分离,截留分子量为3000Da,过膜液喷雾干燥得海参低聚肽粉;所述复合蛋白酶的质量配比为:中性蛋白酶:木瓜蛋白酶:风味蛋白酶=2:5:4。
(4)取花胶低聚肽粉70份、当归粉28份、桃仁粉35份、海参低聚肽粉20份、枸杞粉40份、茯苓粉25份、山药粉30份、酸枣仁粉35份、玫瑰粉20份,于三维运动混合机中混合20min,充分混匀,得到粉剂。
实施例4
一种含有花胶低聚肽的祛斑美容组合物,组分为花胶低聚肽粉80份、当归粉32份、桃仁粉40份、海参低聚肽粉24份、枸杞粉40份、茯苓粉32份、山药粉32份、酸枣仁粉40份、玫瑰粉24份。
制备方法为:
(1)花胶低聚肽粉的制备方法:花胶处理后,加入18倍质量体积的水制成匀浆液置于酶解罐中,然后加入花胶蛋白质质量5%的复合蛋白酶,在50℃下酶解4小时,酶反应pH值控制在8.0,酶解结束后升温至80℃灭酶10分钟,得到花胶蛋白酶解液,将蛋白酶解液8000转/分钟离心10分钟,去除颗粒状物质,然后采用膜分离技术进行分离,截留分子量为3000Da,过膜液喷雾干燥得花胶低聚肽粉;所述复合蛋白酶的质量配比为:中性蛋白酶:胰蛋白酶:菠萝蛋白酶:风味蛋白酶=5:5:3:3。
(2)花胶低聚肽的分离纯化方法:将(1)中的花胶低聚肽粉加水溶解,配制成浓度为100mg/mL的溶液,采用葡聚糖凝胶Sephadex LH-20柱色谱法(3.0×100cm)进行分离纯化,流动相为40%甲醇,流速为0.5mL/min,洗脱液280nm测定吸光度,根据吸光度值收集所需峰;采用高效液相色谱进一步纯化,色谱条件如下:依利特C18色谱柱(4.6mm×250mm,5μm),流动相A为0.06%三氟乙酸水(v/v),流动相B为乙腈,梯度洗脱条件为:0~15min,3%B,15~20min,3%~10%B,20~30min,10%B~20%B,30~40min,20%B~35%B,流速为0.8mL/min,检测波长为280nm,收集保留时间在26分钟的色谱峰,浓缩后冷冻干燥得花胶小分子活性肽。
(3)海参低聚肽粉的制备方法:海参处理后,加入17倍质量体积的水制成匀浆液置于酶解罐中,然后加入海参质量3%的复合蛋白酶,在45℃下酶解4小时,酶反应pH值控制在9.0,酶解结束后升温至80℃灭酶10分钟,得到海参蛋白酶解液,将蛋白酶解液8000转/分钟离心10分钟,去除颗粒状物质,然后采用膜分离技术进行分离,截留分子量为3000Da,过膜液喷雾干燥得海参低聚肽粉;所述复合蛋白酶的质量配比为:中性蛋白酶:木瓜蛋白酶:风味蛋白酶=2:4:4。
(4)取花胶低聚肽粉80份、当归粉32份、桃仁粉40份、海参低聚肽粉24份、枸杞粉40份、茯苓粉32份、山药粉32份、酸枣仁粉40份、玫瑰粉24份,于三维运动混合机中混合18min,充分混匀,得到粉剂。
实施例5
一种含有花胶低聚肽的祛斑美容组合物,组分包括花胶低聚肽粉、当归粉、桃仁粉、海参低聚肽粉、枸杞粉、茯苓粉、山药粉、酸枣仁粉、玫瑰粉。
含有花胶低聚肽的祛斑美容组合物,组分为:花胶低聚肽粉50-100份、当归粉20-40份、桃仁粉30-50份、海参低聚肽粉10-30份、枸杞粉30-50份、茯苓粉20-40份、山药粉20-40份、酸枣仁粉20-50份、玫瑰粉20-30份。
进一步地,该组方的组分为:花胶低聚肽粉65-80份、当归粉26-32份、桃仁粉30-40份、海参低聚肽粉15-24份、枸杞粉34-40份、茯苓粉25-32份、山药粉20-32份、酸枣仁粉25-40份、玫瑰花粉20-24份。
该组方的组分还可以为:花胶低聚肽粉55-90份、当归粉22-36份、桃仁粉33-45份、海参低聚肽粉11-27份、枸杞粉33-45份、茯苓粉22-36份、山药粉22-36份、酸枣仁粉22-45份、玫瑰花粉22-27份。
该组方可以制备为粉剂、颗粒剂、片剂、胶囊剂、丸剂、口服溶液等形式,优选以粉剂、颗粒剂的形式产品。
本发明还涉及到该组方中花胶低聚肽制备、分离纯化,以及海参低聚肽的制备方法:
(1)花胶低聚肽的制备方法:花胶处理后,加入15~20倍质量体积的水制成匀浆液置于酶解罐中,然后加入花胶蛋白质质量3~5%的复合蛋白酶,在50~55℃下酶解3~5小时,酶反应pH值控制在7.0~8.0,酶解结束后升温至80~90℃灭酶10分钟,得到花胶蛋白酶解液,将蛋白酶解液8000转/分钟离心10分钟,去除颗粒状物质,然后采用膜分离技术进行分离,截留分子量为3000Da,过膜液喷雾干燥得花胶低聚肽粉。所述复合蛋白酶的质量配比为:中性蛋白酶:胰蛋白酶:菠萝蛋白酶:风味蛋白酶=(4~6):(3~5):(1~3):(2~4)。
(2)花胶低聚肽的分离纯化方法:将(1)中的花胶低聚肽粉加水溶解,配制成浓度为100mg/mL的溶液,采用葡聚糖凝胶Sephadex LH-20柱色谱法(3.0×100cm)进行分离纯化,流动相为40%甲醇,流速为0.4~0.6mL/min,洗脱液280nm测定吸光度,根据吸光度值收集所需峰;采用高效液相色谱进一步纯化,色谱条件如下:C18色谱柱(4.6mm×250mm,5μm),流动相A为含体积百分数0.06%~0.08%三氟乙酸水,流动相B为乙腈,梯度洗脱条件为:0~15min,3%B;15~20min,3%~10%B;20~30min,10%B~20%B;30~40min,20%B~35%B;流速为0.8mL/min,检测波长为280nm,收集保留时间在26分钟的色谱峰,浓缩后冷冻干燥得花胶小分子活性肽。
(3)海参低聚肽的制备方法:海参处理后,加入15~20倍质量体积的水制成匀浆液置于酶解罐中,然后加入海参质量2~5%的复合蛋白酶,在40~50℃下酶解4小时,酶反应pH值控制在8.0~9.0,酶解结束后升温至80~90℃灭酶10分钟,得到海参蛋白酶解液,将蛋白酶解液8000转/分钟离心10分钟,去除颗粒状物质,然后采用膜分离技术进行分离,截留分子量为3000Da,过膜液喷雾干燥得海参低聚肽粉;所述复合蛋白酶的质量配比为:中性蛋白酶:木瓜蛋白酶:风味蛋白酶=(2~4):(3~5):(3~5)。
其中,步骤(1)中花胶低聚肽90%以上肽的分子量小于1000Da。
步骤(2)得到的花胶小分子活性肽经液相色谱检测为单一峰,利用高效液相色谱-质谱测定结构,其氨基酸序列为:Gly-Pro-Leu-Ala-Gly-Pro。进行酪氨酸酶抑制活性测定,结果显示该小分子肽具有较强酪氨酸酶抑制活性。
步骤(3)中海参低聚肽90%以上肽的分子量小于1000Da。
对比例1
一种含有花胶的祛斑美容组合物,组分为花胶粉90份、当归粉30份、桃仁粉40份、海参低聚肽粉25份、枸杞粉50份、茯苓粉35份、山药粉40份、酸枣仁粉40份、玫瑰粉25份。
制备方法为:
(1)花胶粉的制备方法:将花胶用粉碎机粉碎5min,过80目筛,得到花胶粉。
(2)海参低聚肽粉的制备方法:海参处理后,加入17倍质量体积的水制成匀浆液置于酶解罐中,然后加入海参质量3%的复合蛋白酶,在45℃下酶解4小时,酶反应pH值控制在9.0,酶解结束后升温至80℃灭酶10分钟,得到海参蛋白酶解液,将蛋白酶解液8000转/分钟离心10分钟,去除颗粒状物质,然后采用膜分离技术进行分离,截留分子量为3000Da,过膜液喷雾干燥得海参低聚肽粉;所述复合蛋白酶的质量配比为:中性蛋白酶:木瓜蛋白酶:风味蛋白酶=2:4:4。
(4)取花胶粉90份、当归粉30份、桃仁粉40份、海参低聚肽粉25份、枸杞粉50份、茯苓粉35份、山药粉40份、酸枣仁粉40份、玫瑰粉25份,于三维运动混合机中混合18min,充分混匀,得到粉剂。
对比例2
一种含有花胶低聚肽的祛斑美容组合物,组分为花胶低聚肽粉85份、桃仁粉40份、枸杞粉40份、茯苓粉35份、山药粉30份、酸枣仁粉38份、玫瑰粉20份。
制备方法为:
(1)花胶低聚肽粉的制备方法:花胶处理后,加入17倍质量体积的水制成匀浆液置于酶解罐中,然后加入花胶蛋白质质量3.5%的复合蛋白酶,在50℃下酶解4小时,酶反应pH值控制在7.6,酶解结束后升温至85℃灭酶10分钟,得到花胶蛋白酶解液,将蛋白酶解液8000转/分钟离心10分钟,去除颗粒状物质,然后采用膜分离技术进行分离,截留分子量为3000Da,过膜液喷雾干燥得花胶低聚肽粉;所述复合蛋白酶的质量配比为:中性蛋白酶:胰蛋白酶:菠萝蛋白酶:风味蛋白酶=4:5:2:4。
(2)花胶低聚肽的分离纯化方法:将(1)中的花胶低聚肽粉加水溶解,配制成浓度为100mg/mL的溶液,采用葡聚糖凝胶Sephadex LH-20柱色谱法(3.0×100cm)进行分离纯化,流动相为40%甲醇,流速为0.4mL/min,洗脱液280nm测定吸光度,根据吸光度值收集所需峰;采用高效液相色谱进一步纯化,色谱条件如下:依利特C18色谱柱(4.6mm×250mm,5μm),流动相A为0.07%三氟乙酸水(v/v),流动相B为乙腈,梯度洗脱条件为:0~15min,3%B,15~20min,3%~10%B,20~30min,10%B~20%B,30~40min,20%B~35%B,流速为0.8mL/min,检测波长为280nm,收集保留时间在花胶小分子活性肽。
(3)取花胶低聚肽粉85份、桃仁粉40份、枸杞粉40份、茯苓粉35份、山药粉30份、酸枣仁粉38份、玫瑰粉20份,于三维运动混合机中混合18min,充分混匀,得到粉剂。
实验验证
以本发明实施例2、3、4和对比例1、2得到的产品作为样品,通过动物实验验证其祛斑美容作用。
选取70只18~22g,7周龄的雌性小鼠,随机分成7组(正常对照组,模型组,实验组1、2、3、4、5),每组10只。
模型组、实验组小鼠处理:将小鼠背部剃毛,使皮肤充分暴露,每周1次,剃毛面积为4cm×3cm。将小鼠皮肤在光源20cm处,用中波紫外线局部照射,每日1次,每次30min。后肢肌肉注射黄体酮注射液(20mg·kg-1),两后肢交替注射,每日1次。同时,将小鼠置于小鼠束缚器内束缚,每日1次,每次1h。持续30天,得到肝郁气滞型黄褐斑模型小鼠。
正常对照组小鼠处理:将小鼠在光源20cm处,用中波紫外线局部照射,每日1次,每次30min。后肢肌肉注射生理盐水(20mg·kg-1),两后肢交替注射,每日1次。持续30天。
根据表1要求给各小组小鼠进行灌胃试验,每日一次,连续30天。实验结束后将所有小鼠处死。
表1各组小鼠灌胃实验方案
组别 | 小鼠类别 | 灌胃样品 | 每日剂量 |
正常对照组 | 正常对照组小鼠 | 生理盐水 | 0.2ml/10g |
模型组 | 肝郁气滞型黄褐斑模型小鼠 | 生理盐水 | 0.2ml/10g |
实验组1 | 肝郁气滞型黄褐斑模型小鼠 | 实施例2产品 | 500mg/kg |
实验组2 | 肝郁气滞型黄褐斑模型小鼠 | 实施例3产品 | 500mg/kg |
实验组3 | 肝郁气滞型黄褐斑模型小鼠 | 实施例4产品 | 500mg/kg |
实验组4 | 肝郁气滞型黄褐斑模型小鼠 | 对比例1产品 | 500mg/kg |
试验组5 | 肝郁气滞型黄褐斑模型小鼠 | 对比例2产品 | 500mg/kg |
小鼠皮肤组织检测:1、取适量的小鼠经紫外线照射的皮肤组织,加入10倍体积的冰水,匀浆后离心,检测上清液中的酪氨酸、脂褐质、超氧化物歧化酶(SOD)、丙二醛(MDA)含量。2、取1cm×1cm的小鼠经紫外线照射的皮肤组织观察黑色素细胞的分布情况。
小鼠肝脏组织检测:取适量的小鼠肝脏组织,加入10倍体积的冰水,匀浆后离心,检测上清液中的酪氨酸、超氧化物歧化酶(SOD)、丙二醛(MDA)含量。
其中,采用高效液相法测定酪氨酸,荧光比色法测定脂褐质,黄嘌呤氧化酶法测定SOD,硫代巴比妥酸法测定MDA。用10%的福尔马林固定小鼠皮肤,然后常规石蜡包埋切片,再经脱蜡至水、抗原修复后,用HMB45(melanoma黑素瘤)为第一抗体,以免疫组化方法显示表皮中的黑色素颗粒,来观察黑色素细胞分布。
各组小鼠皮肤组织的实验结果如表2所示。
各组小鼠肝脏组织的实验结果如表3所示。
首先与正常对照组相比,模型组的小鼠皮肤中黑色素细胞数量、酪氨酸、脂褐质、MDA含量明显增加,SOD含量降低,肝脏中的酪氨酸和MDA的含量也显著增加,表示造模成功。
皮肤组织的实验结果表明实验组1、2、3均可以明显减少小鼠皮肤中黑色素细胞的数量,同时降低皮肤中酪氨酸、脂褐质、MDA的含量,而且还能显著提高皮肤中的SOD活性;实验组4、5也能降低小鼠皮肤中黑色素细胞的数量,酪氨酸、脂褐质、MDA含量,提高SOD活性,但是治疗效果没有实验组1、2、3好。
肝脏组织的实验结果表明实验组1、2、3均可以明显减少小鼠肝脏中酪氨酸和MDA的含量,提高皮肤中的SOD活性;实验组4、5也能降低小鼠肝脏中酪氨酸和MDA的含量,对SOD活性有一定的提高,但是治疗效果差于实验组1、2、3。
结论:实验结果表明本发明的组方具有良好的祛斑美容效果,而且实施例2、3、4的组方与对比例1、2的组方相比,效果更好,可以证明本发明组方的原料有协同增效促进的作用。
尽管本发明的描述是通过特定实施例进行的,但本领域技术人员应当理解的是,本发明也可以涵盖于如文中所说的本发明的界限之内的其他实施方式。
表2各组小鼠皮肤组织中黑色素细胞数和酪氨酸、脂褐质、MDA含量及SOD活性
表3各组小鼠肝脏组织中酪氨酸、MDA含量及SOD活性
尽管本发明的描述是通过特定实施例进行的,但本领域技术人员应当理解的是,本发明也可以涵盖如文中所说的本发明的界限之内的其他实施方式。
序列表
<110> 大连深蓝肽科技研发有限公司
<120> 一种含有花胶低聚肽的祛斑美容组合物及其制备方法和用途
<160> 1
<170> SIPOSequenceListing 1.0
<210> 3
<211> 6
<212> PRT
<213> 黄花鱼(Larimichthys)
<400> 3
Gly Pro Leu Ala Gly Pro
1 5
Claims (7)
1.一种含有花胶低聚肽的祛斑美容组合物,其特征在于,包括
花胶低聚肽粉50-100份
当归粉20-40份
桃仁粉30-50份
海参低聚肽粉10-30份
枸杞粉30-50份
茯苓粉20-40份
山药粉20-40份
酸枣仁粉20-50份
玫瑰粉20-30份;
其中,所述的花胶低聚肽粉为花胶小分子活性肽粉,花胶小分子活性肽的氨基酸序列为Gly-Pro-Leu-Ala-Gly-Pro。
2.根据权利要求1所述的美容组合物,其特征在于,花胶低聚肽基于如下方式制备而得:花胶处理后,加入15~20倍质量体积的水制成匀浆液置于酶解罐中,然后加入花胶蛋白质质量3~5%的复合蛋白酶,在50~55℃下酶解3~5小时,酶反应pH值控制在7.0~8.0,酶解结束后升温至80~90℃灭酶10分钟,得到花胶蛋白酶解液,将蛋白酶解液8000转/分钟离心10分钟,去除颗粒状物质,然后采用膜分离技术进行分离,截留分子量为3000Da,过膜液喷雾干燥得花胶低聚肽粉,所述复合蛋白酶的质量配比为:中性蛋白酶:胰蛋白酶:菠萝蛋白酶:风味蛋白酶=(4~6):(3~5):(1~3):(2~4)。
3.根据权利要求2所述的美容组合物,其特征在于,花胶低聚肽中90%以上肽的分子量小于1000Da。
4.根据权利要求3所述的美容组合物,其特征在于,花胶小分子活性肽由花胶低聚肽分离纯化而得:将花胶低聚肽粉加水溶解,配制成浓度为100 mg/mL的溶液,采用葡聚糖凝胶Sephadex LH-20柱色谱法进行分离纯化,流动相为40%甲醇,流速为0.4~0.6 mL/min,洗脱液280nm测定吸光度,根据吸光度值收集所需峰;采用高效液相色谱进一步纯化,色谱条件如下:C18色谱柱,流动相A为含体积百分数 0.06%~0.08%三氟乙酸水,流动相B为乙腈,梯度洗脱条件为:0~15 min,3%B;15~20 min,3%~10%B;20~30 min,10%B~20%B;30~40min,20%B~35%B;流速为0.8mL/min,检测波长为280nm,收集保留时间在26分钟的色谱峰,浓缩后冷冻干燥得花胶小分子活性肽,花胶小分子活性肽氨基酸序列为:Gly-Pro-Leu-Ala-Gly-Pro。
5.根据权利要求1所述的美容组合物,其特征在于,海参低聚肽基于如下方式制备而得:海参处理后,加入15~20倍质量体积的水制成匀浆液置于酶解罐中,然后加入海参质量2~5%的复合蛋白酶,在40~50℃下酶解4小时,酶反应pH值控制在8.0~9.0,酶解结束后升温至80~90℃灭酶10分钟,得到海参蛋白酶解液,将蛋白酶解液8000转/分钟离心10分钟,去除颗粒状物质,然后采用膜分离技术进行分离,截留分子量为3000Da,过膜液喷雾干燥得海参低聚肽粉;所述复合蛋白酶的质量配比为:中性蛋白酶:木瓜蛋白酶:风味蛋白酶=(2~4):(3~5):(3~5)。
6.根据权利要求5所述的美容组合物,其特征在于,海参低聚肽中90%以上肽的分子量小于1000Da。
7.根据权利要求1所述的美容组合物,其特征在于,为粉剂、颗粒剂、片剂、胶囊剂、丸剂、口服溶液中的一种。
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