CN110812306B - 皮肤保养及修复组合物 - Google Patents
皮肤保养及修复组合物 Download PDFInfo
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- CN110812306B CN110812306B CN201810911350.1A CN201810911350A CN110812306B CN 110812306 B CN110812306 B CN 110812306B CN 201810911350 A CN201810911350 A CN 201810911350A CN 110812306 B CN110812306 B CN 110812306B
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- amniotic fluid
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- skin
- egg
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Abstract
本文涉及可用于皮肤保养与修复的羊水和/或其提取物,该羊水来自胚龄为5‑12天的鸡蛋或者来自发育时期与所述胚龄的鸡蛋所处的发育时期相对应的鸡以外的其它禽类的蛋;或来自胎龄为8‑14天的啮齿类动物的胚胎,或来自发育时期与胎龄为8‑14天的啮齿类动物的发育时期相对应的啮齿类动物以外的其它非人哺乳动物的胚胎。该羊水和/或其提取物还可用于延缓皮肤老化、预防皮肤疾病或癌症。
Description
技术领域
本发明涉及皮肤保养及修复,具体涉及皮肤保养及修复组合物。
背景技术
随着人们物质生活水平的提高,皮肤保养越来越引起人们的重视,各种化妆品、护肤品已成为人们生活中必不可少的日常用品。
由于紫外线而产生活性氧是众所周知的。如果活性氧中自由基型活性氧与脂质等氧化性底物进行反应,则会诱发连锁的氧化反应,从而作为自由基的活性氧会增强对皮肤等身体组织的损伤。皮肤由于时常暴露于氧、紫外线,因此是由自由基引起的氧化应激损伤最大的组织。近年来,由于紫外线而产生的各种活性氧引起皮脂和/或脂质的过氧化、蛋白变性、酶抑制等,从而短期时会诱发皮肤炎症等,长期时会成为老化、癌等。此外,活性氧、过氧化脂质还与特应性皮炎、接触性皮炎、银屑病等皮肤疾病有关。因此,活性氧(自由基)与皮肤老化、皮肤疾病密切相关。
具备捕捉自由基的能力的物质可以抑制或停止自由基链反应。因此,可以期待配合了抗氧化剂的皮肤外用剂对由光氧化应激引起的皮肤老化(例如,斑、皱纹、松弛等)具有预防、改善效果。此外,还可以期待作为与自由基相关的各种皮肤疾病用皮肤外用剂的预防、改善效果。
此外,人体因为与外界持续接触,包括呼吸、外界污染、放射线照射等因素,不断地在人体体内产生自由基。科学研究表明,癌症、衰老或其它疾病大都与过量自由基的产生有关联。抗氧化可以有效克服这些自由基所带来的危害。因此,抗氧化是保健品、化妆品企业主要的研发方向之一。
本领域仍然需要抗氧化组合物,用于皮肤保养和修复以及预防癌症和抗衰老。
发明内容
本发明提供一种抗氧化组合物,该组合物含有羊水和/或其提取物,其中,所述羊水来自胚龄为5-12天的鸡蛋,优选胚龄为6-11天的鸡蛋,更优选胚龄为7-9天的鸡蛋,更优选胚龄为7-8天的鸡蛋,或者来自发育时期与所述胚龄的鸡蛋所处的发育时期相对应的鸡以外的其它禽类的蛋;或来自胎龄为8-14天的啮齿类动物的胚胎,或来自发育时期与胎龄为8-14天的啮齿类动物的发育时期相对应的啮齿类动物以外的其它非人哺乳动物的胚胎。
在一个或多个实施方案中,所述提取物在pH5.8-8.0之间不和离子交换柱结合,且其所含成分的分子量在500-1200道尔顿范围。
在一个或多个实施方案中,所述组合物为化妆品或食品。
在一个或多个实施方案中,所述化妆品还含有酯、表面活性剂、保湿剂、水溶性高分子、增稠剂、皮膜剂、紫外线吸收剂、金属离子螯合剂、醇、糖、氨基酸、有机胺、pH调节剂、皮肤营养剂、维生素、抗氧化剂、抗氧化助剂、香料和水中的一种或多种。
在一个或多个实施方案中,所述化妆品为液体、乳液、膏霜类、粉类、块状或油状化妆品,其中,所述液体类化妆品包括但不限于洗面乳、浴液、洗发液、化妆水、香水、洁肤水、精华液和原液;所述乳液类化妆品包括但不限于蜜类、奶类、护发乳和精华乳;所述膏霜类化妆品包括但不限于润面霜、粉底霜、洗发膏、遮瑕膏、精华霜和妆前霜;所述粉类化妆品包括但不限于香粉、爽身粉、散粉、洁肤粉和蜜粉;所述块状化妆品包括但不限于粉饼、口红和发蜡;所述油状化妆品包括但不限于润肤油、润发油和精华油。
在一个或多个实施方案中,所述化妆品包括面膜、洗面奶、化妆水、润肤霜、面部精华液和眼部精华液。
在一个或多个实施方案中,所述食品还含有可食用的成分。
本发明还提供羊水和/或其提取物在制备皮肤保养及修复用的产品中的应用,或在制备预防与自由基产生相关的疾病用的药物中的应用;其中,所述羊水来自胚龄为5-12天的鸡蛋,优选胚龄为6-11天的鸡蛋,更优选胚龄为7-9天的鸡蛋,更优选胚龄为7-8天的鸡蛋,或者来自发育时期与所述胚龄的鸡蛋所处的发育时期相对应的鸡以外的其它禽类的蛋;或来自胎龄为8-14天的啮齿类动物的胚胎,或来自发育时期与胎龄为8-14天的啮齿类动物的发育时期相对应的啮齿类动物以外的其它非人哺乳动物的胚胎。
在一个或多个实施方案中,所述提取物在pH5.8-8.0之间不和离子交换柱结合,且其所含成分的分子量在500-1200道尔顿范围。
在一个或多个实施方案中,皮肤老化为光氧化应激引起的皮肤老化,表现为斑、皱纹和/或松弛。
在一个或多个实施方案中,与自由基产生相关的疾病包括但不限于皮肤疾病、衰老和癌症。
在一个或多个实施方案中,所述皮肤疾病包括由于紫外线而产生的各种活性氧引起皮脂和/或脂质的过氧化、蛋白变性、酶抑制,从而诱发的皮肤炎症,和与活性氧、过氧化脂质相关的特应性皮炎、接触性皮炎或银屑病。
在一个或多个实施方案中,所述癌症包括与过量自由基的产生相关的癌症,如皮肤癌。
在一个或多个实施方案中,所述皮肤保养及修复用的产品为化妆品。
本发明还提供一种抗氧化用药物组合物,该药物组合物为皮肤外用制剂,含有该羊水或提取物,其中,所述羊水来自胚龄为5-12天的鸡蛋,优选胚龄为6-11天的鸡蛋,更优选胚龄为7-9天的鸡蛋,更优选胚龄为7-8天的鸡蛋,或者来自发育时期与所述胚龄的鸡蛋所处的发育时期相对应的鸡以外的其它禽类的蛋;或来自胎龄为8-14天的啮齿类动物的胚胎,或来自发育时期与胎龄为8-14天的啮齿类动物的发育时期相对应的啮齿类动物以外的其它非人哺乳动物的胚胎;优选地,所述提取物在pH5.8-8.0之间不和离子交换柱结合,且其所含成分的分子量在500-1200道尔顿范围。
本发明还提供一种皮肤美容方法,所述方法包括将羊水和/或其提取物或含有该羊水或提取物的化妆品涂抹在皮肤表面的步骤;其中,所述羊水来自胚龄为5-12天的鸡蛋,优选胚龄为6-11天的鸡蛋,更优选胚龄为7-9天的鸡蛋,更优选胚龄为7-8天的鸡蛋,或者来自发育时期与所述胚龄的鸡蛋所处的发育时期相对应的鸡以外的其它禽类的蛋;或来自胎龄为8-14天的啮齿类动物的胚胎,或来自发育时期与胎龄为8-14天的啮齿类动物的发育时期相对应的啮齿类动物以外的其它非人哺乳动物的胚胎;优选地,所述提取物在pH5.8-8.0之间不和离子交换柱结合,且其所含成分的分子量在500-1200道尔顿范围。
附图说明
图1:胚龄为7天的鸡蛋的羊水的HPLC检测结果。
图2:胚龄为11天的鸡蛋的羊水的HPLC检测结果。
图3:胚龄为13天的鸡蛋的羊水的HPLC检测结果。
图4:不同胚龄的鸡蛋羊水的抗自由基能力。其中,横坐标表示胚龄,纵坐标表示清除率。
图5:鸡胚胎成纤维细胞在不同培养条件下的生长曲线。
图6:来自鸡蛋的羊水对对人脐静脉内皮细胞(HUVEC)的生长活力和迁移能力的影响。
图7:来自鸭蛋的羊水对鸡胚胎成纤维细胞的生长活力和迁移能力的影响。其中,横坐标表示培养基,纵坐标表示OD450值。
图8:自鸡蛋的羊水促进原代心肌细胞的生长。其中,横坐标表示培养基,纵坐标表示OD450值。
图9:凝胶柱GE HiLoad 16/600Superdex75pg分离色谱图。
图10:细胞活力检测凝胶柱GE HiLoad 16/600Superdex75pg分离馏分。横坐标表示培养基,其中,FBS表示胎牛血清;DMEM为Dulbecco's Modified Eagle Medium;EE表示羊水;“EE”表示冻干羊水;S-200B表示B峰的馏分;Q UNBOUND表示阴离子柱未结合馏分;3-1到3-6分别表示第三步纯化中等体积的馏分1-6。
图11:细胞活力检测阳离子交换柱GE HiPrep SP和阴离子交换柱HiPrep Q分离得到的未结合馏分。横坐标表示培养基,其中,FBS表示胎牛血清;DMEM为Dulbecco'sModified Eagle Medium;EE表示羊水;“EE”表示冻干羊水;Hiprep SP-UN表示未结合Hiprep SP柱的馏分;Hiprep Q-UN表示未结合Hiprep Q柱的馏分;Hiprep Q-Bound表示结合Hiprep Q柱的馏分。
图12:使用本发明的羊水能明显促进伤口愈合。数据表示为第0天的伤口面积减去第2天或第4天的伤口面积后与第0天的伤口面积之比(%)。
具体实施方式
应理解,在本发明范围中,本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合,从而构成优选的技术方案。
本文中,羊水可来自禽蛋和非人哺乳动物。禽蛋指禽类的蛋。优选的禽类为家禽,如鸡、鸭和鹅。优选的是,本发明使用胚龄在5-20天、优选6-15天的禽蛋。应理解,不同禽蛋,合适的胚龄未必相同。例如,当使用鸡蛋时,优选使用胚龄为5-12天的鸡蛋,更优选使用胚龄为6-11天的鸡蛋,更优选使用胚龄为7-9天的鸡蛋、更优选使用胚龄为7-8天的鸡蛋。当使用其它禽类的蛋时,可使用其发育时期与上述胚龄的鸡蛋所处的发育时期相对应的蛋。例如,当使用鸭蛋时,胚龄为8-10天、尤其是8-9天的鸭蛋可能是最好的。
可采用常规的方法获得禽蛋羊水。例如,可敲击相应胚龄的蛋的钝端,使蛋壳碎裂,将蛋壳剥开形成一个直径约为2厘米的口子。然后用镊子小心撕开壳膜和卵黄膜,注意不要破坏羊膜。将包裹着胚胎的羊膜和相连组织从壳中倾倒至培养皿中,用注射器刺入羊膜抽取羊水,直至羊膜紧贴胚胎,由此即可获得用于本发明的羊水。
本文中,羊水还可来自非人哺乳动物,尤其是啮齿类动物,如来自小鼠。其它非人哺乳动物可以是常见的家畜,例如牛、羊、狗、猫、猪等。在某些实施方案中,羊水来自胎龄为8-14天的啮齿类动物的胚胎,或来自其发育时期与胎龄为8-14天的啮齿类动物所处的发育时期相对应的非人哺乳动物的胚胎。可采用常规的方法获得羊水。例如,用手术剪剪开怀孕8-14天的小鼠腹腔,小心取出并剪开子宫,用注射器刺入羊膜抽取羊水,直至羊膜紧贴胚胎,由此即可获得用于本发明的羊水。
应理解,必要时,可对羊水进行离心,以分离出可能含有的杂质,例如卵黄等,尽可能获得纯的羊水。离心后获得的上清液即为用于本发明的羊水。应理解的是,获取羊水的所有步骤都需在无菌条件下进行;另外,本文所示的“羊水”应指“纯”的羊水,即分离自禽蛋或非人哺乳动物胚胎的不含有禽蛋内或非人哺乳动物胚胎内其它成分、且也未被外源物质污染的羊水。纯的羊水可储存于-60℃以下的冰箱中,解冻后再使用。
在某些实施方案中,本发明使用羊水的提取物。优选地,该提取物在pH5.8-8.0之间不和离子交换柱子结合,且其所含成分的分子量在500-1200道尔顿范围内。可从羊水中分离出分子量为500-1200道尔顿的中性馏分,由此获得所述提取物。可采用本领域周知的凝胶柱和离子交换柱来实施本文的方法。例如,可使用周知的凝胶层析柱(如下文所述的各种凝胶层析柱)从羊水中分离出分子量为500-1200道尔顿的馏分,然后使用离子交换方法(如使用下文所述的离子交换柱)从该馏分中分离出中性馏分。或者,也可先用离子交换方法(如使用下文所述的离子交换柱)从羊水中分离出中性馏分,然后再使用凝胶层析柱(如下文所述的各种凝胶层析柱)分离出该中性馏分中分子量在500-1200道尔顿范围内的馏分。
在某些实施方案中,可先从羊水中分离得到分子量在500-2000道尔顿的中性馏分,然后再从中分离得到分子量在500-1200道尔顿范围内的馏分。具体而言,该方法可包括以下步骤:
(1)从羊水中分离得到分子量为500-2000道尔顿的中性馏分;和
(2)从该分子量为500-2000道尔顿的中性馏分中分离得到分子量为500-1200道尔顿的中性馏分。
步骤(1)可通过使用凝胶层析和离子交换方法来实现。通过凝胶层析柱分离出羊水中分子量在500-2000道尔顿的成分,而通过离子交换可获得不带电荷(中性)的馏分。
本文中,可采用各种市售的凝胶层析柱来实施凝胶层析,这类凝胶层析柱包括但不限于GE公司的SephacrylS-100、SephacrylS-200、SephacrylS-300、Sephacryl S-400、Superose 12、Superose 6、Superdex 12和Superdex 6等。应理解,也可使用分离范围为500-10000道尔顿的其它任意凝胶层析填料。通常,在使用凝胶层析柱时,可先用ddH2O平衡凝胶层析柱,流速可根据实际情况确定。例如,在某些实施方案中,流速可以是0.5-50ml/min,如1ml/min。通常,紫外吸收在200-300nm之间,如280nm。待紫外吸收曲线平稳,回归基线后,结束平衡。平衡结束后,可上样。上样流速根据实际制备情况确定。上样结束后,可用脱气ddH2O洗脱粗品,收集分子量在500-2000道尔顿之间的馏分。需要时,可重复凝胶层析的分离数次,混合每一次分离时相同出峰时间的馏分。
本文中,可采用本领域周知的方法将带电荷的成分与不带电荷的成分分离。例如,可使用离子交换方法实现。阴离子交换和阳离子交换都可用于本发明方法中。在某些实施方案中,本文采用阴离子交换法。可使用市售的阴离子交换柱,包括但不限于GE公司的DEAESepharose、ANX Sepharose、Q Sepharose、Capto DEAE、Capto Q、Mono Q和Mini Q等。应理解,也可使用其它品牌的阴离子交换填料。或者,也可使用市售的阳离子交换柱,包括但不限于CM Sepharose、SP Sepharose、Capto S、Mono S和Mini S等。
通常,在实施离子交换时,先用缓冲液平衡离子交换柱。缓冲液可以是本领域常规的缓冲液,例如可使用磷酸盐缓冲液,尤其是磷酸钠缓冲液。缓冲液的pH可根据所使用的离子交换柱确定。例如,使用阴离子交换柱时,可使用pH为7.5~8.5、优选7.5~8.0的缓冲液平衡阴离子交换柱;使用阳离子交换柱时,可使用pH为5.8~7.0、优选5.8~6.5的缓冲液平衡阳离子交换柱。在某些实施方案中,该磷酸钠缓冲液含有Na2HPO4和NaH2PO4,pH为约5.8或8.0。本发明优选使用阴离子交换柱进行分离。流速可根据实际情况确定。例如,在某些实施方案中,流速可以是0.5-50ml/min,如1ml/min。通常,待280nm紫外吸收曲线平稳,回归基线后,结束平衡。平衡结束后,可上样,收集流出部分(即未与柱结合的部分)。上样流速根据实际制备情况确定。
步骤(1)中,可先进行凝胶层析,分离出分子量在500-2000道尔顿的馏分,然后再进行离子交换,分离出中性馏分;或者,也可以先进行离子交换,分离出羊水中的中性馏分,然后再通过凝胶层析将中性馏分中分子量在500-2000道尔顿范围内的活性成分分离出来,获得分子量在500-2000道尔顿之间的中性馏分。
步骤(2)中的主要目的是进一步对步骤(1)获得的中性馏分进行分离,获得分子量大小在500-1200道尔顿范围内的活性成分。本文中,可使用市售的凝胶层析柱来分离得到分子量在500-1200道尔顿范围内的馏分。合适的凝胶层析柱包括但不限于GE公司的HiLoadSuperdex 16/600Superdex75pg、Superdex Peptide、Superdex 200和Superdex 30等。应理解,也可以使用分离范围在500-10000道尔顿的其它品牌的凝胶层析填料。
通常,可先用ddH2O平衡凝胶柱,流速可根据实际情况确定。例如,在某些实施方案中,流速可以是0.5-50ml/min,如1ml/min。通常,待280nm紫外吸收曲线平稳,回归基线后,结束平衡。平衡结束后,可上样。上样流速根据实际制备情况确定。上样结束后,可用脱气ddH2O洗脱粗品,收集馏分,获得所含成分的分子量在500-1200道尔顿范围的馏分,即为本文所述的提取物。
采用上述方法获得的提取物,将其配制成pH为5.8-8.0的溶液后过多种离子交换柱(包括DEAE Sepharose、Q Sepharose、Mono Q、CM Sepharose、SP Sepharose和Mono S),其所含活性成分均不与这些离子交换柱结合。
本文所述的羊水和/或其提取物具有抗氧化作用,因此可用于皮肤保养及修复,延缓皮肤老化,抗衰老。因此,在某些实施方案中,本文提供一种抗氧化组合物,该组合物含有本文所述的羊水和/或其提取物。组合物中羊水和/或其提取物的含量可根据该抗氧化组合物的具体用途以及该组合物所含其它成分的含量由本领域技术人员容易地确定。举例而言,抗氧化组合物中本发明羊水和/或其提取物的含量通常为抗氧化物组合物种类的0.05-20wt%,如0.1-15wt%、0.1-1wt%不等。当使用羊水提取物时,其用量可以稍低一些,例如可以是0.05-5wt%,如0.1-2wt%。在某些实施方案中,该抗氧化组合物为化妆品组合物,除含有所述羊水和/或其提取物外,还可含有化妆品组合物中通常可含有的其它成分,包括辅料和其它的化妆品活性成分。
例如,可以根据化妆品的不同种类而适当配合其它辅料成分,包括但不限于酯、阴离子表面活性剂、阳离子表面活性剂、两性表面活性剂、非离子表面活性剂、保湿剂、水溶性高分子、增稠剂、皮膜剂、紫外线吸收剂、金属离子螯合剂、低级醇、多元醇、糖、氨基酸、有机胺、高分子乳液、pH调节剂、皮肤营养剂、维生素、抗氧化剂、抗氧化助剂、香料和水等之中的一种或多种,按照目的剂型通过常规方法来制备。
可列举的阴离子表面活性剂包括但不限于脂肪酸皂(例如月桂酸钠、棕榈酸钠等),高级烷基硫酸酯盐(例如月桂基硫酸钠、月桂基硫酸钾等),烷基醚硫酸酯盐(例如POE-月桂基硫酸三乙醇胺、POE-月桂基硫酸钠等),N-酰基肌氨酸(例如月桂酰基肌氨酸钠等),高级脂肪酸酰胺磺酸盐(例如N-肉豆蔻酰基-N-甲基牛磺酸钠、椰子油脂肪酸甲基牛磺酸钠、月桂基甲基牛磺酸钠等),磷酸酯盐(POE-油基醚磷酸钠、POE-硬脂基醚磷酸等),磺基琥珀酸盐(例如二-2-乙基己基磺基琥珀酸钠、单月桂酰基单乙醇酰胺聚氧乙烯磺基琥珀酸钠、月桂基聚丙二醇磺基琥珀酸钠等),烷基苯磺酸盐(例如线性十二烷基苯磺酸钠、线性十二烷基苯磺酸三乙醇胺、线性十二烷基苯磺酸等),高级脂肪酸酯硫酸酯盐(例如氢化椰子油脂肪酸甘油硫酸钠等),N-酰基谷氨酸盐(例如N-月桂酰基谷氨酸单钠、N-硬脂酰基谷氨酸二钠、N-肉豆蔻酰基-L-谷氨酸单钠等),磺化油(例如土耳其红油等),POE-烷基醚羧酸,POE-烷基烯丙基醚羧酸盐,α-烯烃磺酸盐,高级脂肪酸酯磺酸盐,仲醇硫酸酯盐,高级脂肪酸烷醇酰胺硫酸酯盐,月桂酰基单乙醇酰胺琥珀酸钠,N-棕榈酰基天冬氨酸双三乙醇胺,酪蛋白钠等。
可列举的阳离子表面活性剂包括但不限于烷基三甲基铵盐(例如硬脂基三甲基氯化铵、月桂基三甲基氯化铵等),烷基吡啶盐(例如鲸蜡基氯化吡啶等),二硬脂基二甲基氯化铵、二烷基二甲基铵盐,氯化聚(N,N’-二甲基-3,5-亚甲基哌啶),烷基季铵盐,烷基二甲基苄基铵盐,烷基异喹啉盐,POE-烷基胺,烷基胺盐,多胺脂肪酸衍生物,戊醇脂肪酸衍生物,苯扎氯铵,苄索氯铵等。
可列举的两性表面活性剂包括但不限于咪唑啉类两性表面活性剂(例如2-十一烷基-N,N,N-(羟乙基羧甲基)-2-咪唑啉钠、2-椰子油基-2-氢氧化咪唑啉-1-羧基乙氧基二钠盐等),甜菜碱类表面活性剂(例如2-十七烷基-N-羧甲基-N-羟乙基咪唑啉甜菜碱、月桂基二甲基氨基乙酸甜菜碱、烷基甜菜碱、酰胺甜菜碱、磺基甜菜碱等)等。
可列举的亲油性非离子表面活性剂包括但不限于脱水山梨糖醇脂肪酸酯类(例如脱水山梨糖醇单油酸酯、脱水山梨糖醇单异硬脂酸酯、脱水山梨糖醇单月桂酸酯、脱水山梨糖醇单棕榈酸酯、脱水山梨糖醇单硬脂酸酯、脱水山梨糖醇倍半油酸酯、脱水山梨糖醇三油酸酯、五-2-乙基己酸二甘油脱水山梨糖醇酯、四-2-乙基己酸二甘油脱水山梨糖醇酯等),甘油聚甘油脂肪酸酯类(例如单棉籽油脂肪酸甘油酯、单芥酸甘油酯、倍半油酸甘油酯、单硬脂酸甘油酯、α,α’-油酸焦谷氨酸甘油酯、单硬脂酸甘油苹果酸酯等),丙二醇脂肪酸酯类(例如单硬脂酸丙二醇酯等),氢化蓖麻油衍生物,甘油烷基醚等。
可列举的亲水性非离子表面活性剂包括但不限于POE-脱水山梨糖醇脂肪酸酯类(例如POE-脱水山梨糖醇单油酸酯、POE-脱水山梨糖醇单硬脂酸酯、POE-脱水山梨糖醇单油酸酯、POE-脱水山梨糖醇四油酸酯等),POE-山梨糖醇脂肪酸酯类(例如POE-山梨糖醇单月桂酸酯、POE-山梨糖醇单油酸酯、POE-山梨糖醇五油酸酯、POE-山梨糖醇单硬脂酸酯等),POE-甘油脂肪酸酯类(例如POE-甘油单硬脂酸酯、POE-甘油单异硬脂酸酯、POE-甘油三异硬脂酸酯等POE-单油酸酯等),POE-脂肪酸酯类(例如POE-二硬脂酸酯、POE-单二油酸酯、二硬脂酸乙二醇酯等),POE-烷基醚类(例如POE-月桂基醚、POE-油基醚、POE-硬脂基醚、POE-二十二烷基醚、POE-2-辛基十二烷基醚、POE-胆甾烷醇醚等),普流尼克(Pluronic)型类,POE-蓖麻油氢化蓖麻油衍生物(例如POE-蓖麻油、POE-氢化蓖麻油、POE-氢化蓖麻油单异硬脂酸酯、POE-氢化蓖麻油三异硬脂酸酯、POE-氢化蓖麻油单焦谷氨酸单异硬脂酸二酯、POE-氢化蓖麻油马来酸等),烷醇酰胺(例如椰子油脂肪酸二乙醇酰胺、月桂酸单乙醇酰胺、脂肪酸异丙醇酰胺等),POE-丙二醇脂肪酸酯,POE-烷基胺,POE-脂肪酸酰胺,蔗糖脂肪酸酯,烷基乙氧基二甲基氧化胺,三油基磷酸等。
可列举的保湿剂包括但不限于聚乙二醇、丙二醇、甘油、1,3-丁二醇、木糖醇、山梨糖醇、麦芽糖醇、硫酸软骨素、透明质酸、硫酸粘多糖、胆甾醇基-12-羟基硬脂酸酯、乳酸钠、胆汁酸盐、dl-吡咯烷酮羧酸盐、氧化烯衍生物、短链可溶性胶原、刺梨提取物、欧蓍草提取物、草木樨提取物等。
可列举的天然的水溶性高分子包括但不限于植物系高分子(例如阿拉伯胶、黄蓍胶、半乳聚糖、瓜尔胶、角豆树胶、刺梧桐树胶、角叉菜聚糖、果胶、琼脂、榅桲籽、淀粉(大米、玉米、马铃薯、小麦)、甘草酸),微生物类高分子(例如黄原胶、葡聚糖、琥珀酰葡聚糖、支链淀粉等),动物类高分子(例如胶原、酪蛋白、白蛋白、明胶等)等。
可列举的半合成的水溶性高分子包括但不限于淀粉类高分子(例如羧甲基淀粉、甲基羟丙基淀粉等),纤维素类高分子(甲基纤维素、乙基纤维素、甲基羟丙基纤维素、羟乙基纤维素、纤维素硫酸钠、羟丙基纤维素、羧甲基纤维素、羧甲基纤维素钠、结晶纤维素、纤维素粉末等),藻酸类高分子(例如藻酸钠、藻酸丙二醇酯等)等。
可列举的合成的水溶性高分子包括但不限于乙烯基类高分子(例如聚乙烯醇、聚乙烯基甲基醚、聚乙烯吡咯烷酮、羧基乙烯基聚合物等),聚氧乙烯类高分子(例如聚乙二醇20000、40000、600000的聚氧乙烯聚氧丙烯共聚物等),丙烯酸类高分子(例如聚丙烯酸钠、聚丙烯酸乙酯、聚丙烯酰胺等),聚乙烯亚胺,阳离子聚合物等。
可列举的增稠剂包括但不限于阿拉伯胶、角叉菜聚糖、刺梧桐树胶、黄蓍胶、角豆树胶、榅桲籽、酪蛋白、糊精、明胶、果胶酸钠、藻酸钠、甲基纤维素、乙基纤维素、CMC、羟乙基纤维素、羟丙基纤维素、PVA、PVM、PVP、聚丙烯酸钠、羧基乙烯基聚合物、刺槐豆胶、瓜尔胶、罗望子胶、二烷基二甲基铵硫酸纤维素、黄原胶、硅酸铝镁、膨润土、锂蒙脱石、硅酸酐等。
可列举的紫外线吸收剂包括但不限于苯甲酸类紫外线吸收剂(例如对氨基苯甲酸(以下简称为PABA)、PABA单甘油酯、N,N-二丙氧基PABA乙酯、N,N-二乙氧基PABA乙酯、N,N-二甲基PABA乙酯、N,N-二甲基PABA丁基酯、N,N-二甲基PABA乙酯等),邻氨基苯甲酸类紫外线吸收剂(高基-N-乙酰基邻氨基苯甲酸酯等),水杨酸类紫外线吸收剂(例如水杨酸戊酯、水杨酸基酯、水杨酸高基酯、水杨酸辛酯、水杨酸苯酯、水杨酸苄酯、对异丙醇苯基水杨酸酯等),肉桂酸类紫外线吸收剂(例如辛基甲氧基肉桂酸酯、乙基-4-异丙基肉桂酸酯、甲基-2,5-二异丙基肉桂酸酯、乙基-2,4-二异丙基肉桂酸酯、甲基-2,4-二异丙基肉桂酸酯、丙基对甲氧基肉桂酸酯、异丙基对甲氧基肉桂酸酯、异戊基对甲氧基肉桂酸酯、辛基对甲氧基肉桂酸酯(2-乙基己基对甲氧基肉桂酸酯)、2-乙氧基乙基对甲氧基肉桂酸酯、环己基对甲氧基肉桂酸酯、乙基-α-氰基-β-苯基肉桂酸酯、2-乙基己基-α-氰基-β-苯基肉桂酸酯、甘油基单-2-乙基己酰基-二对甲氧基肉桂酸酯等),二苯甲酮类紫外线吸收剂(例如2,4-二羟基二苯甲酮、2,2’-二羟基-4-甲氧基二苯甲酮、2,2’-二羟基-4,4’-二甲氧基二苯甲酮、2,2’,4,4’-四羟基二苯甲酮、2-羟基-4-甲氧基二苯甲酮、2-羟基-4-甲氧基-4’-甲基二苯甲酮、2-羟基-4-甲氧基二苯甲酮-5-磺酸盐、4-苯基二苯甲酮、2-乙基己基-4’-苯基-二苯甲酮-2-羧酸酯、2-羟基-4-正辛氧基二苯甲酮、4-羟基-3-羧基二苯甲酮等),3-(4’-甲基苯亚甲基)-d,l-樟脑、3-苯亚甲基-d,l-樟脑,2-苯基-5-甲基苯并唑,2,2’-羟基-5-甲基苯基苯并三唑,2-(2’-羟基-5’-叔辛基苯基)苯并三唑,2-(2’-羟基-5’-甲基苯基苯并三唑),二茴香酰基甲烷,4-甲氧基-4’-叔丁基二苯甲酰基甲烷,二吗啉代基哒嗪酮,2-乙基己基-2-氰基-3,3-二苯基丙烯酸酯,2,4-双-[[4-(2-乙基己氧基)-2-羟基]苯基]-6-(4-甲氧基苯基)-(1,3,5)-三嗪等。
可列举的金属离子螯合剂包括但不限于1-羟基乙烷-1,1-二膦酸、1-羟基乙烷-1,1-二膦酸四钠盐、依地酸二钠、依地酸三钠、依地酸四钠、柠檬酸钠、多磷酸钠、偏磷酸钠、葡萄糖酸、磷酸、柠檬酸、抗坏血酸、琥珀酸、依地酸、乙二胺羟基乙基三乙酸三钠等。
可列举的低级醇包括但不限于乙醇、丙醇、异丙醇、异丁醇、叔丁醇等。
可列举的多元醇包括但不限于二元醇(例如乙二醇、丙二醇、1,3-丙二醇、1,2-丁二醇、1,3-丁二醇、1,4-丁二醇、2,3-丁二醇、1,5-戊二醇、2-丁烯-1,4-二醇、己二醇、辛二醇等),三元醇(例如甘油、三羟甲基丙烷等),四元醇(例如1,2,6-己三醇等季戊四醇等),五元醇(例如木糖醇等),六元醇(例如山梨醇、甘露醇等),多元醇聚合物(例如二甘醇、双丙甘醇、三甘醇、聚丙二醇、四甘醇、二甘油、聚乙二醇、三甘油、四甘油、聚甘油等),二元醇烷基醚类(例如乙二醇单甲基醚、乙二醇单乙基醚、乙二醇单丁基醚、乙二醇单苯基醚、乙二醇单己基醚、乙二醇单2-甲基己基醚、乙二醇异戊基醚、乙二醇苄基醚、乙二醇异丙基醚、乙二醇二甲基醚、乙二醇二乙基醚、乙二醇二丁基醚等),二元醇烷基醚类(例如二甘醇单甲基醚、二甘醇单乙基醚、二甘醇单丁基醚、二甘醇二甲基醚、二甘醇二乙基醚、二甘醇丁基醚、二甘醇甲基乙基醚、三甘醇单甲基醚、三甘醇单乙基醚、丙二醇单甲基醚、丙二醇单乙基醚、丙二醇单丁基醚、丙二醇异丙基醚、二丙二醇甲基醚、二丙二醇乙基醚、二丙二醇丁基醚等),二元醇醚酯(例如乙二醇单甲基醚乙酸酯、乙二醇单乙基醚乙酸酯、乙二醇单丁基醚乙酸酯、乙二醇单苯基醚乙酸酯、乙二醇二己二酸酯、乙二醇二琥珀酸酯、二甘醇单乙基醚乙酸酯、二甘醇单丁基醚乙酸酯、丙二醇单甲基醚乙酸酯、丙二醇单乙基醚乙酸酯、丙二醇单丙基醚乙酸酯、丙二醇单苯基醚乙酸酯等),甘油单烷基醚(例如鲨油醇、鲨肝醇等),糖醇(例如山梨醇、麦芽糖醇、麦芽三糖、甘露醇、蔗糖、赤藓醇、葡萄糖、果糖、淀粉分解糖、麦芽糖、木糖、淀粉分解糖还原醇等),四氢糠醇,POE-四氢糠醇,POP-丁基醚,POP·POE-丁基醚,三聚氧丙烯甘油醚,POP-甘油醚,POP-甘油醚磷酸,POP·POE-季戊四醇醚、聚甘油等。
可列举的单糖包括但不限于三碳糖(例如D-甘油醛、二羟基丙酮等),四碳糖(例如D-赤藓糖、D-赤藓酮糖、D-苏糖、赤藓糖醇等),五碳糖(例如L-阿拉伯糖、D-木糖、L-来苏糖、D-阿拉伯糖、D-核糖、D-核酮糖、D-木酮糖、L-木酮糖等),六碳糖(例如D-葡萄糖、D-塔罗糖、D-阿洛酮糖、D-半乳糖、D-果糖、L-半乳糖、L-甘露糖、D-塔格糖等),七碳糖(例如庚醛糖、庚糖等),八碳糖(例如辛酮糖等),脱氧糖(例如2-脱氧-D-核糖、6-脱氧-L-半乳糖、6-脱氧-L-甘露糖等),氨基糖(例如D-葡糖胺、D-半乳糖胺、唾液酸、氨基糖醛酸、胞壁酸等),糖醛酸(例如D-葡糖醛酸、D-甘露糖醛酸、L-古洛糖醛酸、D-半乳糖醛酸、L-艾杜糖醛酸等)等。
可列举的低聚糖包括但不限于蔗糖、龙胆三糖、伞形糖、乳糖、车前糖、异剪秋罗糖类,海藻糖、蜜三糖、剪秋罗糖类、水苏糖、毛蕊花糖类等。
可列举的多糖包括但不限于纤维素、榅桲籽、硫酸软骨素、淀粉、半乳聚糖、硫酸皮肤素、糖原、阿拉伯胶、硫酸乙酰肝素、透明质酸、黄蓍胶、硫酸角质素、软骨素、黄原胶、硫酸粘多糖、瓜尔胶、葡聚糖、硫酸角质、刺槐豆胶、琥珀酰葡聚糖、栝楼仁酸等。
可列举的氨基酸包括但不限于中性氨基酸(例如苏氨酸、半胱氨酸等),碱性氨基酸(例如羟基赖氨酸等)等。此外,作为氨基酸衍生物,可以举出例如酰基肌氨酸钠(月桂酰基肌氨酸钠)、酰基谷氨酸盐、酰基β-丙氨酸钠、谷胱甘肽、吡咯烷酮羧酸等。
可列举的有机胺包括但不限于单乙醇胺、二乙醇胺、三乙醇胺、吗啉、三异丙醇胺、2-氨基-2-甲基-1,3-丙二醇、2-氨基-2-甲基-1-丙醇等。
可列举的高分子乳液包括但不限于丙烯酸树脂乳液、聚丙烯酸乙酯乳液、丙烯酸树脂液、聚丙烯酸烷基酯乳液、聚乙酸乙烯酯树脂乳液、天然橡胶胶乳等。
可列举的pH调节剂包括但不限于乳酸-乳酸钠、柠檬酸-柠檬酸钠、琥珀酸-琥珀酸钠等缓冲剂等。
可列举的维生素类包括但不限于维生素A、B1、B2、B6、C、E及其衍生物、泛酸及其衍生物、生物素等。
可列举的抗氧化剂包括但不限于生育酚类、二丁基羟基甲苯、丁基羟基茴香醚、没食子酸酯类等。
可列举的抗氧化助剂包括但不限于磷酸、柠檬酸、抗坏血酸、马来酸、丙二酸、琥珀酸、富马酸、脑磷脂、六偏磷酸盐、植酸、乙二胺四乙酸等。
组合物中还可含有防腐剂(对羟基苯甲酸乙酯、对羟基苯甲酸丁酯等),消炎剂(例如甘草酸衍生物、甘草次酸衍生物、水杨酸衍生物、日柏醇、氧化锌、尿囊素等),美白剂(例如胎盘提取物、虎耳草提取物、熊果苷等),各种提取物(例如黄柏、黄连、紫根、芍药、当药、桦木、鼠尾草、枇杷、人参、芦荟、锦葵、鸢尾、葡萄、薏苡仁、丝瓜、百合、藏红花、川芎、生姜、小连翘、洋葱、大蒜、辣椒、陈皮、当归、海藻等),赋活剂(例如蜂王浆料、感光素、胆甾醇衍生物等),促进血液循环剂(例如壬酸香草酰胺)、烟酸苄酯、烟酸β-丁氧基乙酯、辣椒素、姜油酮、斑蝥酊、鱼石脂、鞣酸、α-龙脑、烟酸生育酚酯、六烟酸肌醇酯、环扁桃酯、桂利嗪、苯苄唑啉、乙酰胆碱、戊脉安、风箱树苷素、γ-谷维素等),抗脂溢剂(例如硫磺、二甲噻蒽等),抗炎剂(例如凝血酸、硫基牛磺酸、亚牛磺酸等)等。
进一步地,还可以适当配合依地酸二钠、依地酸三钠、柠檬酸钠、多磷酸钠、偏磷酸钠、葡萄糖酸、苹果酸等金属离子螯合剂,咖啡因、鞣酸、异博定、凝血酸及其衍生物,甘草、木瓜、鹿蹄草等的各种生药提取物,乙酸生育酚酯、甘草酸、甘草酸及其衍生物或其盐等药物,维生素C、抗坏血酸磷酸镁、抗坏血酸葡糖苷、熊果苷、曲酸等美白剂,精氨酸、赖氨酸等氨基酸及其衍生物,果糖、甘露糖、赤藓醇、海藻糖、木糖醇等糖类等。
更多的辅料可参见CN102395351A,本文将其全部内容以引用的方式纳入本文。
本发明的化妆品可是液体、乳液、膏霜类、粉类、块状或油状。液体类化妆品包括但不限于洗面乳、浴液、洗发液、化妆水、香水、洁肤水、精华液、原液等;乳液类化妆品包括但不限于蜜类、奶类、护发乳、精华乳;膏霜类化妆品包括但不限于润面霜、粉底霜、洗发膏、遮瑕膏、精华霜、妆前霜;粉类化妆品包括但不限于香粉、爽身粉、散粉、洁肤粉、蜜粉;块状化妆品包括但不限于粉饼、化妆盒、口红和发蜡;油状化妆品包括但不限于润肤油、润发油和精华油等。可根据化妆品的不同用途选择合适的辅料。辅料的用量为其常规用量。可根据不同的化妆品类型,采用常规的制备方法制备得到相应的化妆品。在某些实施方案中,所述化妆品包括面膜、洗面奶、化妆水、润肤霜、面部精华液和眼部精华液。
在某些实施方案中,本发明的抗氧化组合物可以是食品。食品中除含有本文所述的羊水和/或其提取物外,还可含有本领域周知的各种可食用的产品。例如,食品可以是饮品,其含有饮品中常规添加的成分,如糖、水、各种添加剂等以及本文所述的羊水和/或其提取物。在某些实施方案中,食品是保健品,还有本领域已知的其它营养成分,如氨基酸、各种维生素、植物蛋白等。换言之,可在目前市售的各种可食用的产品中添加本文所述的羊水和/或其提取物。对食品中所述羊水和/或其提取物的含量并无特殊限制,例如可在0.1-90%的范围内,甚至更高。食用本发明的食品可起到消除体内自由基的作用。
在某些实施方案中,所述抗氧化用产品为皮肤保养及修复用产品,可用于延缓或改善皮肤老化。该产品可以是本文所述外用的化妆品或内服的食品(如保健品)。通常,皮肤老化为光氧化应激引起的皮肤老化,通常表现为斑、皱纹和/或松弛。在某些实施方案中,所述抗氧化用产品为预防或改善与自由基产生相关的疾病的产品,同样地,该产品也可以是外用产品或内服产品。与自由基产生相关的疾病包括但不限于皮肤疾病、衰老和癌症。皮肤疾病包括由于紫外线而产生的各种活性氧引起皮脂和/或脂质的过氧化、蛋白变性、酶抑制等,从而短期时会诱发皮肤炎症,也包括与活性氧、过氧化脂质相关的特应性皮炎、接触性皮炎、银屑病等皮肤疾病。不受任何理论的限制,通常认为,衰老过程中的退行性变化是由于细胞正常代谢过程中产生的自由基的有害作用造成的。生物体的衰老过程是机体的组织细胞不断产生的自由基积累结果。本领域已知某些自由基抑制剂及抗氧化剂可以延长细胞和动物的寿命。体内自由基防御能力随年龄的增长而减弱。此外,衰老是由新陈代谢减退而引起的。本文所述的羊水和/或其提取物能清除自由基,促进细胞生长,因此可用于预防和/或延缓衰老进程。本文中,癌症尤其包括那些与过量自由基的产生相关的癌症,如皮肤癌。
在某些实施方案中,本发明还提供一种抗氧化用的药物组合物,该药物组合物为皮肤外用制剂,含有本文所述的羊水或其添加剂,和任选的适用于皮肤外用的药学上可接受的载体,包括植物或矿物油、白矿油(白软石蜡)、支链脂肪或油脂、动物脂肪和高分子醇(大于C12)。优选的载体是活性成分能溶解在其中的那些载体。也可包括乳化剂、稳定剂、保湿剂和抗氧化剂,以及如果需要的话,给予颜色或香味的试剂。此外,这些外用制剂可包含透皮渗透增强剂。这种增强剂的例子可参见美国专利号3,989,816和4,444,762。本发明的外用制剂可通过优选合适的载体来制成油剂、霜剂、乳液剂、药膏等。
皮肤美容方法也包括在本文所述的范围之内,该方法包括将羊水和/或其提取物或含有该羊水或提取物的化妆品涂抹在皮肤表面的步骤。应理解,该方法可以仅仅是美容方法,不以治疗为目的。
下文将以具体实施例的方式阐述本发明。应理解,这些实施例仅仅是阐述性的,并不意图限制本发明的范围。实施例中所用到的方法、试剂和仪器,除非另有说明,否则为本领域常规的方法、试剂和仪器。
实施例一
1、材料
a)仪器和工具
微电脑全自动孵化器(正大TMZF880),洁净培养皿,1.0ml注射器(江西洪达TM),经70%酒精消毒的镊子,不锈钢筛子,无菌离心管(#SCT-50ML-25-S)和低速冷冻离心机(中佳KDC-2046)。
b)试剂和生物材料
胚龄7天的鸡蛋。
2、实验流程
取鸡蛋,敲击朝上放置的、较为扁平的钝端,使蛋壳碎裂,将蛋壳剥开形成一个直径约为2厘米的口子,边缘应尽量平整。用镊子小心撕开壳膜和卵黄膜,注意不要破坏羊膜。观察胚胎发育状况,只有发育良好且符合对应阶段标准的胚胎可用于抽提羊水。
将包裹着胚胎的羊膜和相连组织从壳中倾倒至培养皿中,用注射器刺入羊膜抽取羊水,针口斜面应背对胚胎,直至羊膜紧贴胚胎,然后将澄清、无色、无异物的羊水注入冰盒内的离心管中。
用镊子取出羊膜中的胚胎,收集于放置在冰上的不锈钢筛子中,每隔一小时将收集的胚胎用搅拌机匀质化,封装在无菌的塑料储物罐中,倾斜放置于-80℃冰箱中。冷冻后可竖直放置。
通过美谱达TM1800紫外分光光度仪测试收集的羊水抽提液,光度仪标准操作流程参见使用手册。
将收集羊水抽提液的离心管配平后用中佳TM KDC-2046低速冷冻离心机于5℃,3500rpm离心21分钟(离心机标准操作流程参见使用手册)。将上清液倾析转移至洁净的塑料储物罐中,储存于-80℃冰箱中。每批次预留5ml小样用于后续测试。
所有步骤都在无菌条件下进行。
实施例二
本实施例使用日立Primaide型高效液相色谱仪检测不同胚龄的鸡蛋的羊水成分。按该色谱仪的使用说明书进行检测。其中,检测开始前先用100%乙腈洗涤30分钟,流速时间为0.8mL/min,之后用水平衡30分钟,流速0.8mL/min时间。抽取25μL样品并排除气泡,点击色谱仪自带的软件的“数据采集”按钮,选择“方法2”,点击屏幕下方“单分析开始”,待系统出现“等待进样”时,开始注射样品,注射要迅速,注射完后切换阀门。该方法2如下:
时间(min) | 水(%) | 乙腈(%) | 流量(mL/min) |
0.0 | 100.0 | 0.0 | 0.8 |
11.0 | 100.0 | 0.0 | 0.8 |
17.0 | 95.0 | 5.0 | 0.8 |
30.0 | 90.0 | 10.0 | 0.8 |
45.0 | 55.0 | 45.0 | 0.8 |
60.0 | 0.0 | 100.0 | 0.8 |
70.0 | 0.0 | 100.0 | 0.8 |
本实施例检测了胎龄为7天、11天和13天的羊水,结果如图1-3所示。
实施例三
DPPH即1,1-二苯基-2-苦基肼基自由基,其结构如下所示:
DPPH的分子中,由于存在多个吸电子的-NO2和苯环的大π键,所以,氮自由基能稳定存在。
当DPPH自由基被清除,其最大吸收波长519nm处的吸光度A值随之减小。DPPH这种稳定的自由基为清除自由基活性的检测提供了一个理想而又简单的药理模型。本实施例利用DPPH来检测来自鸡胚羊水的抗自由基能力。
取0.8mg的DPPH,溶于20mL溶剂甲醇中,超声5min,充分振摇,使上下各部分均匀。取1mL该DPPH溶液,在519nm处测A0值,A=0.5-0.7。该DPPH溶液避光保存,3.5小时内用完。
采用实施例一所述的方法分别获得胚龄为6天、7天、8天、9天、10天和11天的鸡蛋的羊水,离心后保存在4℃冰箱中待用。
以维生素C作为正对照,测定标准曲线。取不同体积的0.04mg/ml Vc样品加入0.6ml的DPPH,加入无水乙醇补足至1ml,混匀,以甲醇为对照调零,测定519nm波长下的吸光值。重复三次数据后作图。
取不同胚龄的羊水400ul至试管中,加入600ul中配制好的DPPH的甲醇溶液,混匀,反应10min,使气泡不再产生(测定前要混匀)。以甲醇为对照调零,测定519nm的吸光度。
各组的加样信息如下表所示:
实验组 | 样品液 | 95%乙醇(或无水乙醇) | DPPH测试液 | 总体积 |
空白组 | 0mL | 0.4mL | 0.6mL | 1mL |
Vc | n uL | (400-n)uL | 0.6mL | 1mL |
样品组 | 0.4mL | 0mL | 0.6mL | 1mL |
采用以下公司肌酸清除率(抑制率):
清除率(%)=(A0-A)/A0×100%。
结果如图4所示。
实施例四
本实施例测试实施例一的鸡蛋羊水(EE)对鸡胚胎成纤维细胞在不同培养条件下的生长的影响。本实施例使用的DMEM培养基的组成如下:#Cat.11960077,加入1%L-谷氨酰胺(#G0200)和5%FBS(#Cat.10099141)),0.25%胰酶(杭州科易生物TM#CY003),PBS(BITM#02-024-1ACS),0.4%台盼蓝染剂(BBITM#72-57-1)。
1、鸡胚胎成纤维细胞的获得和培养
取胚龄7天的鸡蛋的胚胎,用PBS冲洗胚胎表面,将液体用移液枪吸干净。取出胚胎内脏,将其余组织剪碎至无肉眼可见的大颗粒、团块。加1ml的0.25%胰酶,用枪头使之与组织混匀,将悬液吸取至15ml离心管。用1ml的0.25%胰酶冲洗培养皿,将悬液吸取至同一支15ml离心管。将离心管放入37℃水浴,消化5-7分钟后,加入8ml的DMEM培养基(含PBS)中和胰酶。将离心管放入离心机,离心5-10秒。取出离心管,收集上清液。2000rpm离心该离心上清液2min。弃上清,加入4ml的DMEM培养基,用枪头使细胞重悬。分别取1ml细胞悬液注入10cm细胞培养皿,再加入10ml的DMEM培养基。十字方向晃动培养皿,每个方向至少20次,使细胞分布均匀。于37℃、5%CO2条件下培养。当细胞覆盖70%-90%的培养皿底部时,将细胞传代。
将培养皿从培养箱取出,收集原有的培养基于离心管中。小心加入5ml的PBS清洗细胞。之后,加入500ul、0.25%胰酶,将培养皿放入培养箱,消化1分钟。轻轻拍打培养皿侧边加快消化过程,待细胞团块快速分解,大部分细胞呈漂浮状态,迅速加入9.5ml回收的原有培养基中和胰酶。用移液管吹打培养皿底部,将尽量多的细胞悬液收集至15ml离心管中,于2000rpm离心3min。弃上清,加入4ml的DMEM培养基,用枪头使细胞重悬。分别将1ml细胞悬液注入含有10ml含不同体积比的羊水的新鲜培养基的10cm细胞培养皿中。十字方向晃动培养皿,每个方向至少20次,使细胞分布均匀,于37℃、5%CO2条件下培养。
取生长良好的鸡胚胎成纤维细胞,收集原有的培养基于离心管中。小心加入5ml的PBS清洗细胞,注意不可造成细胞层破损,轻轻晃动后倒除PBS。加入100ul的0.25%胰酶消化2-5分钟(24孔板),用100ul培养基中和。用枪头使之成为单细胞悬液。按照一定倍数稀释该单细胞悬液,加入等量0.4%台盼蓝染液染色,稀释倍数以稀释后细胞数在20-200之间为宜。吸取适量(15ul)细胞悬液,从盖玻片上下边缘加样到血球计数板上,显微镜下计活细胞数。计算活细胞总数,调整细胞浓度至1×105个细胞/ml。每24小时取样一次,每次取3个孔细胞,进行常规胰酶消化、制备单细胞悬液、显微镜计数。以时间(天)为横轴,细胞浓度为纵轴绘制生长曲线。细胞个数=细胞总计数/4×104×稀释倍数,细胞浓度=该细胞个数/ml。
结果如图5所示。图5显示,在共培育96小时以后,添加EE的实验组中鸡胚胎成纤维细胞的数量明显高于与未添加EE的对照的细胞数量。
实施例五
采用与实施例一相同的方法获得胚龄为8天的鸭蛋的羊水。采用划痕实验测试鸡蛋羊水对鸡胚胎成纤维细胞和鸭蛋羊水对人脐静脉内皮细胞(HUVEC)的生长活力和迁移能力的影响。鸭蛋羊水获自胚龄8天的鸭蛋,采用实施例一的方法获得。鸡胚胎成纤维细胞采用实施例五所述的方法获得,人脐静脉内皮细胞从市售途径获得。
本实施例使用的DMEM培养基的组成如下:#Cat.11960077,加入1%L-谷氨酰胺(#G0200)和5%FBS(#Cat.10099141)),0.25%胰酶(杭州科易生物TM#CY003),PBS(BITM#02-024-1ACS),0.4%台盼蓝染剂(BBITM#72-57-1)。
实验前第一天,准备6孔板,用马克笔在6孔板背后用直尺画出5-6条分布均匀的横线,横穿过孔;再在中线位置画一条垂直的竖线以指示划痕的位置。在每个孔中加入约5×105个处于对数生长期的细胞,原则上接种过夜后融合率达到90%。
实验当天,用枪头比着直尺,沿马克笔竖线,垂直于6孔板底面划线。尽量不要倾斜、弯曲,不同孔之间最好用同一支枪头,宽度在1000-2000um为宜。每个孔用2ml PBS清洗3次,洗去划痕处的细胞。各个孔中分别加入2ml含不同含量的EE的培养基,常规培养,每48小时换液。从划痕计时为0h,每24小时定点拍照,测量划痕两侧细胞间距。观察每个孔中细胞生长状况;以时间(天)为横轴,每个孔中划痕距离为纵轴绘制图表;计算每个孔中划痕愈合的速度。
结果如图6和7所示。图6显示来自鸡蛋的羊水对对人脐静脉内皮细胞(HUVEC)的生长活力和迁移能力的影响,添加5%(体积比)的羊水明显对HUVEC的愈合具有非常明显的促进作用。图7显示来自鸭蛋的羊水对鸡胚胎成纤维细胞的生长活力和迁移能力的影响,羊水的添加对鸡胚胎成纤维细胞的愈合也显示出非常明显的促进作用。
实施例六
1、分离原代心肌细胞(VM)
取乳鼠心室于预冷PBS清洗,之后在DMEM/F12中剪碎心脏组织。37℃水浴震荡,用0.04%胶原酶Ⅱ+0.08%胰酶消化。用筛网将已经消化下来的细胞过滤离心,1000r/min,10min。加15%FBS细胞培养液铺板,在5%CO2饱和湿度37℃培养箱培养。
2、细胞活力检测
将原代心肌细胞消化后,铺于96孔板中,6000个/孔,每一组五个复孔。在5%CO2饱和湿度37℃培养箱培养24小时之后,分别用培养基DMEM/F12、含10%FBS的DMEM/F12、含10%FBS和5%EE的DMEM/F12代替原来培养基15%FBS的DMEM/F12。培养48小时后,每孔加入10μl CCK-8试剂。孵育2小时后,于酶标仪在450nm检测吸收值。
结果如图8所示。
实施例七
本实施例的目的在于通过分析柱凝胶柱SephacrylS-200、阴离子交换柱HiPrepQ、脱盐柱HiPrep 26/10Desalting、HiLoad 16/600Superdex75pg逐步纯化鸡胚羊水中具有生物活性的化合物。
1、材料
1.1纯化样品:新鲜的胚龄为7天的鸡蛋羊水,50ml。
1.2主要实验设备及耗材
1)GE AKTA purifier;
2)凝胶柱GE Sephacryl S-200;
3)阴离子交换柱GEHiPrep Q;
4)脱盐柱GEHiPrep 26/10Desalting;
5)凝胶柱GEHiLoad 16/600Superdex75pg;
6)Superloop 10ml。
2、方法
2.1溶液制备
(1)磷酸钠缓冲液A(50mM Na2HPO4+NaH2PO4,pH 8.0)的制备:
46.6ml 1mol/l Na2HPO4与3.4ml 1mol/l NaH2PO4混合,加ddH2O定容至1L。
(2)磷酸钠缓冲液B(50mM Na2HPO4+NaH2PO4与500mM NaCl,pH 8.0)的制备:
46.6ml 1mol/l Na2HPO4与3.4ml 1mol/l NaH2PO4和250ml 2mol/l NaCl混合,加ddH2O定容至1L。
2.2实验方法
2.2.2样本处理:新鲜羊水50ml,加入适量己烷,2500rpm、4℃离心20min,获得水相,0.22μm滤膜过滤。
2.2.3样品纯化
第一步:凝胶柱GE Sephacryl S-200
ddH2O平衡凝胶柱:流速2ml/min,直到280nm紫外吸收曲线平稳,回归基线;
上样:流速1ml/min,上样量10ml;
洗脱:用脱气ddH2O洗脱粗品,流速2ml/min,等体积收集馏分,3ml/管。2柱体积(240ml)洗脱;
重复分离纯化5次,每一次中相同出峰时间的部分充分混合;
第二步:阴离子交换柱GE HiPrep Q
磷酸钠缓冲液A(50mM Na2HPO4+NaH2PO4,pH 8.0)平衡阴离子交换柱:流速2ml/min,直到280nm紫外吸收曲线平稳,回归基线;
上样:取第一步纯化后具有生物活性的部分,用泵上样流速1.5ml/min,上样量250ml,同时等体积收集阴离子柱未结合部分,2ml/管;
脱盐:将离子柱中结合和不结合的馏分分别用GE HiPrep 26/10Desalting置换到脱气ddH2O中,收集脱盐后的部分;
第三步:凝胶柱GE HiLoad 16/600Superdex75pg
ddH2O平衡凝胶柱:流速1ml/min,直到280nm紫外吸收曲线平稳,回归基线;
上样:流速1ml/min,上样量10ml;
洗脱:用脱气ddH2O洗脱样品,流速1ml/min,等体积收集馏分,2ml/管。洗脱1.5柱体积(240ml);
测细胞活性:将长势较好的AC16消化后,铺于96孔板中,8000个/孔,每一组五个复孔。在5%CO2饱和湿度37℃培养箱培养2小时,细胞贴壁。用培养基DMEM饥饿培养24小时后,替换成10%FBS的DMEM、DMEM和含20%馏分的培养基。培养24小时后,每孔加入10μl CCK-8试剂。孵育2小时后,于酶标仪在450nm检测吸收值。
3、实验结果
经凝胶柱GE HiLoad 16/600Superdex75pg分离的未结合部分的色谱图如图9所示。细胞活力检测跟踪到具有生物活性的生长因子群,结果如图10所示。
实施例八
采用与实施例七相同的方法,实施以下分离纯化:
1、活性成分分离纯化
第一步:凝胶柱GE Sephacryl S-200
ddH2O平衡凝胶柱:流速2ml/min,直到280nm紫外吸收曲线平稳,回归基线;
上样:流速1ml/min,上样量10ml;
洗脱:用脱气ddH2O洗脱粗品,流速2ml/min,收集分子量在500-2000道尔顿范围内的馏分;
重复分离纯化5次,每一次中相同出峰时间的部分充分混合;
第二步:阳离子交换柱GE HiPrep SP
磷酸钠缓冲液A(50mM Na2HPO4+NaH2PO4,pH 5.8)平衡阳离子交换柱:流速2ml/min,直到280nm紫外吸收曲线平稳,回归基线;
上样:取第一步获得的分子量在500-2000道尔顿范围内的馏分,用泵上样流速1.5ml/min,上样量250ml,收集阳离子交换柱未结合部分;
第三步:凝胶柱GE HiLoad 16/600Superdex75pg
ddH2O平衡凝胶柱:流速1ml/min,直到280nm紫外吸收曲线平稳,回归基线;
上样:取第二步获得的未结合部分上样,流速1ml/min,上样量10ml;
洗脱:用脱气ddH2O洗脱样品,流速1ml/min,收集分子量在500-1200道尔顿范围内的馏分。
2、活性成分检测
将长势较好的AC16消化后,铺于96孔板中,8000个/孔,每一组五个复孔。在5%CO2饱和湿度37℃培养箱培养2小时,细胞贴壁。用培养基DMEM饥饿培养24小时后,替换成10%FBS的DMEM、DMEM和含20%馏分的培养基。培养24小时后,每孔加入10μlCCK-8试剂。孵育2小时后,于酶标仪在450nm检测吸收值。阳离子交换柱GE HiPrep SP处理后不结合区的细胞活力如图11所示。
实施例九
采用与实施例七相同的方法实施以下分离纯化:
1、活性成分分离纯化
第一步:离子交换柱,可使用阴离子交换柱HiPrep Q,使各溶液的pH分别为5.8和8.0,然后分别上样与离子交换柱,流速2ml/min,直到280nm紫外吸收曲线平稳,回归基线;
上样:取羊水,用泵上样流速1.5ml/min,上样量50ml,收集离子柱未结合馏分;
第二步:凝胶柱GE Sephacryl S-200
ddH2O平衡凝胶柱:流速2ml/min,直到280nm紫外吸收曲线平稳,回归基线;
上样:样品为第一步未结合馏分,流速1ml/min,上样量10ml;
洗脱:用脱气ddH2O洗脱粗品,流速2ml/min,收集分子量在500-2000道尔顿范围内的馏分;
第三步:凝胶柱GEHiLoad 16/600Superdex75pg
ddH2O平衡凝胶柱:流速1ml/min,直到280nm紫外吸收曲线平稳,回归基线;
上样:取第二步获得的500-2000道尔顿范围内的馏分上样,流速1ml/min,上样量10ml;
洗脱:用脱气ddH2O洗脱样品,流速1ml/min,收集分子量在500-1200道尔顿范围内的馏分。
2、活性成分检测
将长势较好的AC16消化后,铺于96孔板中,8000个/孔,每一组五个复孔。在5%CO2饱和湿度37℃培养箱培养2小时,细胞贴壁。用培养基DMEM饥饿培养24小时后,替换成10%FBS的DMEM、DMEM和含20%馏分的培养基。培养24小时后,每孔加入10μl CCK-8试剂。孵育2小时后,于酶标仪在450nm检测吸收值。阴离子交换柱GE HiPrepQ处理后不结合区的细胞活力如图11所示。
实施例十
本实施例利用小鼠全层皮肤损伤模型(Murine full-thickness wound model)研究本文所述的羊水和/或其提取物对小鼠伤口愈合的情况。
1、材料
麻醉药(5.00%水合氯醛),75%酒精,实施例一获得的无菌EE,PBS,小鼠(C57BL/6,8周龄)。
2、实验流程
(1)准备9个干净的笼子,将9只相同周龄的成年(6-8周及以上)雌性健康小鼠分别称重后放到笼内。
(2)配置5.00%浓度的水合氯醛,以0.07ml/10g比例用1ml注射器经腹腔注射到小鼠体内(麻醉诱导时间大约5-10分钟,麻醉维持时间大约35分钟)。
(3)待小鼠进入麻醉状态(翻正反射消失后),用小鼠解剖板固定其躯体,用电动剃毛器刮去脖颈和背部毛发,用75%酒精擦拭皮肤消毒。
(4)用一只手食、拇指拎起小鼠颈背部皮肤形成足够面积的褶皱,将皮肤褶皱放在解剖板上;另一手持活检针(孔径6mm),对准褶皱中部施压并旋转至活检处两侧皮肤被打穿脱落。
(5)若有皮肤残留,用镊子和眼科剪清理切开的圆柱状组织块边缘,沿着皮肤活检针留下的印记剪去全层皮肤,创造两个一样的创口。
(6)用体视显微镜拍摄伤口照片,并测量伤口面积,记录数据。伤口应尽量与水平线平行,否则测量误差较大。
(7)将质检合格的100ul EE和PBS(空白对照组)分别用移液枪滴涂于对应小鼠伤口上,用辅料覆盖伤口。
(8)饲养盒中用医用棉取代木屑,单独饲养,防止小鼠刮蹭伤口。此后每隔24*n小时检查并测量伤口,记录照片和数据。
(9)计算比较不同辅料对伤口愈合速率的影响。
3、实验结果
如图12所示,分别于第2、4天测量小鼠伤口面积,两组伤口面积均有减小,EE组愈合速率较高,且无疤痕形成。
实施例十一
修复面膜制作步骤如下:
1、用烧杯称取卡波940后用少量水预溶2小时,待完全溶解后,用配制好的氢氧化钠水溶液(浓度1:10)逐滴滴定至pH到中性;
2、其余原料单独搅拌2小时左右后,慢慢倒入滴定后的卡波溶液中,边倒边搅,混匀后放置冰箱一晚即可。
面膜的配方(质量百分比)如下:
实施例十二
按以下配方制备眼部精华液(质量百分比):
实施例十三
按以下配方制备面部精华液(质量百分比):
Claims (27)
1.一种抗氧化组合物,其特征在于,所述抗氧化组合物含有羊水的提取物,其中,所述羊水来自胚龄为5-11天的鸡蛋或者来自发育时期与所述胚龄的鸡蛋所处的发育时期相对应的鸭蛋,所述提取物在pH5.8-8.0之间不和离子交换柱结合,且其所含成分的分子量在500-1200道尔顿范围。
2.如权利要求1所述的抗氧化组合物,其特征在于,所述羊水来自胚龄为6-11天的鸡蛋。
3.如权利要求1所述的抗氧化组合物,其特征在于,所述羊水来自胚龄为7-9天的鸡蛋。
4.如权利要求1所述的抗氧化组合物,其特征在于,所述羊水来自胚龄为7-8天的鸡蛋。
5.如权利要求1-4中任一项所述的抗氧化组合物,其特征在于,所述组合物为化妆品。
6.如权利要求5所述的抗氧化组合物,其特征在于,所述化妆品还含有表面活性剂、保湿剂、增稠剂、紫外线吸收剂、金属离子螯合剂、pH调节剂、维生素、抗氧化剂、抗氧化助剂、香料和水中的一种或多种。
7.如权利要求6所述的抗氧化组合物,其特征在于,所述化妆品含有酯、醇、糖、氨基酸和有机胺中的一种或多种。
8.如权利要求5所述的抗氧化组合物,其特征在于,所述化妆品含有皮肤营养剂。
9.如权利要求5所述的抗氧化组合物,其特征在于,所述化妆品为液体、膏霜类或粉类化妆品。
10.如权利要求8所述的抗氧化组合物,其特征在于,所述皮肤营养剂为水溶性高分子。
11.如权利要求9所述的抗氧化组合物,其特征在于,
所述液体类化妆品选自洗面乳、浴液、洗发液、化妆水、香水和洁肤水;
所述膏霜类化妆品选自洗发膏、遮瑕膏、精华霜和妆前霜;
所述粉类化妆品选自洁肤粉和蜜粉。
12.如权利要求5所述的抗氧化组合物,其特征在于,
所述化妆品为乳液类化妆品,选自护发乳和精华乳;或
所述化妆品为块状化妆品,选自粉饼、口红和发蜡;或
所述化妆品为精华油。
13.如权利要求5所述的抗氧化组合物,其特征在于,所述化妆品包括面膜、洗面奶、化妆水、润肤霜、面部精华液和眼部精华液。
14.羊水提取物在制备皮肤保养及修复用的产品中的应用;其中,所述羊水来自胚龄为5-11天的鸡蛋或者来自发育时期与所述胚龄的鸡蛋所处的发育时期相对应的鸭蛋,所述提取物在pH5.8-8.0之间不和离子交换柱结合,且其所含成分的分子量在500-1200道尔顿范围。
15.如权利要求14所述的应用,其特征在于,所述羊水来自胚龄为6-11天的鸡蛋。
16.如权利要求14所述的应用,其特征在于,所述羊水来自胚龄为7-9天的鸡蛋。
17.如权利要求14所述的应用,其特征在于,所述羊水来自胚龄为7-8天的鸡蛋。
18.如权利要求14所述的应用,其特征在于,所述皮肤保养及修复用的产品用于延缓或改善光氧化应激引起的皮肤老化,表现为斑、皱纹和/或松弛。
19.如权利要求14所述的应用,其特征在于,所述皮肤保养及修复用的产品为化妆品。
20.一种抗氧化用药物组合物,该药物组合物为皮肤外用制剂,含有羊水的提取物,其中,所述羊水来自胚龄为5-11天的鸡蛋或者来自发育时期与所述胚龄的鸡蛋所处的发育时期相对应的鸭蛋;所述提取物在pH5.8-8.0之间不和离子交换柱结合,且其所含成分的分子量在500-1200道尔顿范围。
21.如权利要求20所述的抗氧化用药物组合物,其特征在于,所述羊水来自胚龄为6-11天的鸡蛋。
22.如权利要求20所述的抗氧化用药物组合物,其特征在于,所述羊水来自胚龄为7-9天的鸡蛋。
23.如权利要求20所述的抗氧化用药物组合物,其特征在于,所述羊水来自胚龄为7-8天的鸡蛋。
24.一种皮肤美容方法,其特征在于,所述方法包括将羊水的提取物或含有该羊水的提取物的化妆品涂抹在皮肤表面的步骤;
其中,所述羊水来自胚龄为5-11天的鸡蛋或者来自发育时期与所述胚龄的鸡蛋所处的发育时期相对应的鸭蛋;所述提取物在pH5.8-8.0之间不和离子交换柱结合,且其所含成分的分子量在500-1200道尔顿范围。
25.如权利要求24所述的皮肤美容方法,其特征在于,所述羊水来自胚龄为6-11天的鸡蛋。
26.如权利要求24所述的皮肤美容方法,其特征在于,所述羊水来自胚龄为7-9天的鸡蛋。
27.如权利要求24所述的皮肤美容方法,其特征在于,所述羊水来自胚龄为7-8天的鸡蛋。
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