CN114146133A - 宽叶苔草在制备具有抗衰老功效产品中的应用 - Google Patents
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Abstract
宽叶苔草在制备具有抗衰老功效产品中的应用，涉及宽叶苔草新用途领域，其中，宽叶苔草为宽叶苔草乙酸乙酯提取物，其制备方法为：取宽叶苔草加75％乙醇浸泡；水浴加热回流提取；用绢布滤出提取液，合并回收溶剂；将提取液置于水浴锅上蒸至无醇味；加入乙酸乙酯，振摇萃取，合并有机层，回收溶剂，水层蒸至无试剂味后进行下一次萃取；回收溶剂，获得浸膏；用热水重新溶解浸膏，过滤后即得宽叶苔草乙酸乙酯提取物。本发明通过试验证实宽叶苔草乙酸乙酯提取物显著增强了SOD和GPX的表达，显著抑制了SABG的表达；增强了成纤维细胞HSF胶原蛋白的产生且抑制了基质金属蛋白酶‑1的表达。由此证明宽叶苔草乙酸乙酯提取物具有抗衰老功效。
Description
技术领域
本发明涉及宽叶苔草新用途技术领域，具体涉及一种宽叶苔草在制备具有抗衰老功效产品中的应用。
背景技术
衰老是一种不可抗拒的自然规律，是每个有生命的个体生长发育的必经阶段，常表现为结构的退行性变化和机能的衰退。衰老可以分为两类：一类是生理性衰老，指机体在成熟期后出现的生理退化过程，主要体现在各器官组织功能随年龄增长而发生的退行性变化；二类是病理性衰老，即因患某些疾病或受外界因素的影响，而加速衰老的过程。
2013年已有学者在《Cell》杂志提出衰老的9个重要因素，这些因素又可以根据其与衰老的关系密切程度分为三级，其中基因组不稳定、端粒缩短、表观遗传学改变、蛋白稳定性改变为一级因素，对营养物质的反馈失衡、线粒体功能障碍、细胞衰老为二级因素，干细胞衰竭、细胞通讯发生变化为三级因素。
衰老过程是不可逆的，但一些药物可以延缓及预防衰老。例如公开号为CN106109362A的中国专利公开了一种抗衰老组合物、抗衰老化妆品及其制备方法，它是通过将车前草籽提取物、羽扇豆提取物以及荷花提取物复配使用起到改善皮肤老化、紧致皮肤等作用。
宽叶苔草常被作为园林景观植物，用作乔木下及低湿处的地被。2017年西北农林科技大学的郭恩辉将宽叶苔草提取物作为植物源抑菌物质，证明了宽叶苔草提取物对多种病原微生物有抑制作用。
目前，宽叶苔草提取物在抗衰老方面的应用尚未见报道。
发明内容
本发明的目的是提供一种宽叶苔草在制备具有抗衰老功效产品中的应用。
本发明为解决技术问题所采用的技术方案如下：
本发明的宽叶苔草在制备具有抗衰老功效产品中的应用。
作为优选的实施方式，所述宽叶苔草为宽叶苔草乙酸乙酯提取物。
作为更优选的实施方式，所述宽叶苔草乙酸乙酯提取物按照以下方法制备：
①称取宽叶苔草，加4-6倍量75％乙醇浸泡10-14h；
②水浴加热回流提取2次，每次1.5-2.5h；用绢布滤出提取液，合并回收溶剂；
③将回收完溶剂的提取液置于水浴锅上蒸至无醇味；
④按提取液与乙酸乙酯的体积比为1:1的比例向上述提取液中加入乙酸乙酯，振摇萃取4-6次，合并有机层，回收溶剂，其中，水层置于水浴锅蒸至无试剂味后再进行下一次萃取；
⑤经萃取获得正水层部分，回收溶剂，获得浸膏；
⑥用40-70℃热水重新溶解浸膏，经0.22μm滤膜过滤后即得宽叶苔草乙酸乙酯提取物。
作为更优选的实施方式，步骤①中，加5倍量75％乙醇浸泡12h。
作为更优选的实施方式，步骤②中，水浴加热回流提取2次，每次2h。
作为更优选的实施方式，步骤④中，振摇萃取5次。
作为更优选的实施方式，步骤⑥中，用温度为50℃的热水重新溶解浸膏。
作为优选的实施方式，所述宽叶苔草乙酸乙酯提取物的作用浓度范围为：0.01-1mg/ml。
作为更优选的实施方式，所述宽叶苔草乙酸乙酯提取物的浓度为：0.825mg/ml。
作为优选的实施方式，所述产品包括：食品、药品、保健品和化妆品。
本发明的有益效果是：
本发明通过试验证实宽叶苔草乙酸乙酯提取物显著增强了抗衰老超氧化物歧化酶SOD和谷胱甘肽过氧化物酶GPX的表达，同时显著抑制了β-半乳糖苷酶SABG的表达；并增强了成纤维细胞HSF胶原蛋白的产生且抑制了成纤维细胞HSF基质金属蛋白酶-1(MMP-1)的表达。由此证实，宽叶苔草乙酸乙酯提取物具有调节抗氧化相关基因表达的作用，从而达到抗衰老的功效。
本发明所提供的宽叶苔草乙酸乙酯提取物在制备具有抗衰老功效产品中的应用开拓了宽叶苔草的新用途，为开发新型的抗衰老抗氧化功效产品提供了新的思路。
附图说明
图1为实施例3中宽叶苔草乙酸乙酯提取物抗衰老作用试验结果。
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楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，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。
试验材料
宽叶苔草，来源于吉林大学天然药物研究中心。
人表皮细胞Hacat，购买自上海酶研生物科技有限公司。
成纤维细胞HSF，购买自上海酶研生物科技有限公司。
细胞裂解液Cell Lysis Buffer，购买自Cell Signaling Technology。
实施例1宽叶苔草提取物的制备
①称取宽叶苔草250g，加5倍量75％乙醇浸泡12h。
②水浴加热回流提取2次，每次2h；用绢布滤出提取液，合并回收溶剂。
③将回收完溶剂的提取液置于水浴锅上蒸至无醇味，提取液终体积控制在500ml左右。
④向上述提取液中加入乙酸乙酯500ml，振摇萃取5次，合并有机层，回收溶剂，其中，水层置于水浴锅蒸至无试剂味后再进行下一次萃取。
同时，以正己烷、氯仿、水饱和正丁醇这三种有机溶剂做平行试验，试验过程同上。
⑤经过上述萃取获得正水层部分，回收溶剂，获得浸膏。
⑥用50℃热水重新溶解浸膏，过0.22μm滤膜，即得宽叶苔草提取物，即宽叶苔草乙酸乙酯提取物、宽叶苔草正己烷提取物、宽叶苔草氯仿提取物、宽叶苔草水饱和正丁醇提取物。
实施例2功效筛选试验
(1)细胞给药
①用含有10％胎牛血清的DMEM(高糖)培养人表皮细胞Hacat，待细胞生长至80-90％密度时消化并传至96孔板。
②24h后待细胞生长至70-80％密度时，将实施例1中经过滤后的各宽叶苔草提取物分别用含10％胎牛血清的DMEM培养液稀释至1.65mg/ml、0.825mg/ml、0.413mg/ml、0.206mg/ml、0.103mg/ml、0.052mg/ml共6个不同浓度并加入人表皮细胞Hacat继续培养。
③继续培养24h后，加入细胞裂解液Cell Lysis Buffer，充分裂解细胞。
(2)功效筛选
24h后收集细胞提取总RNA，通过基因芯片技术检测转录组变化，通过生物信息分析由药物作用引起的表皮细胞基因变化及涉及的生物学功能，结果发现只有宽叶苔草乙酸乙酯提取物具有上调超氧化物歧化酶SOD、谷胱甘肽过氧化物酶GPX和抑制β-半乳糖苷酶SABG的作用，因此，初步推测宽叶苔草乙酸乙酯提取物具有抗衰老功效。
实施例3效果验证性试验
以人表皮细胞Hacat和成纤维细胞HSF为模型，分别以不同浓度宽叶苔草乙酸乙酯提取物作用于人表皮细胞Hacat后，qPCR检测其衰老相关酶(超氧化物歧化酶SOD、谷胱甘肽过氧化物酶GPX、β-半乳糖苷酶SABG)的表达变化；同时，检测宽叶苔草乙酸乙酯提取物作用于成纤维细胞HSF后，胶原蛋白、基质金属蛋白酶-1(MMP-1)的表达变化。其中，阴性对照采用含10％胎牛血清的DMEM做相同处理。检测结果如表1和图1所示。
表1检测结果
由表1和图1可知，宽叶苔草乙酸乙酯提取物显著增强了抗衰老超氧化物歧化酶SOD和谷胱甘肽过氧化物酶GPX的表达，同时显著抑制了β-半乳糖苷酶SABG的表达；并增强了成纤维细胞HSF胶原蛋白的产生且抑制了成纤维细胞HSF基质金属蛋白酶-1(MMP-1)的表达。
综上，宽叶苔草乙酸乙酯提取物具有调节抗氧化相关基因表达的作用，从而达到抗衰老的作用。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.宽叶苔草在制备具有抗衰老功效产品中的应用。
2.根据权利要求1所述的应用，其特征在于，所述宽叶苔草为宽叶苔草乙酸乙酯提取物。
3.根据权利要求2所述的应用，其特征在于，所述宽叶苔草乙酸乙酯提取物按照以下方法制备：
①称取宽叶苔草，加4-6倍量75％乙醇浸泡10-14h；
②水浴加热回流提取2次，每次1.5-2.5h；用绢布滤出提取液，合并回收溶剂；
③将回收完溶剂的提取液置于水浴锅上蒸至无醇味；
④按提取液与乙酸乙酯的体积比为1:1的比例向上述提取液中加入乙酸乙酯，振摇萃取4-6次，合并有机层，回收溶剂，其中，水层置于水浴锅蒸至无试剂味后再进行下一次萃取；
⑤经萃取获得正水层部分，回收溶剂，获得浸膏；
⑥用40-70℃热水重新溶解浸膏，经0.22μm滤膜过滤后即得宽叶苔草乙酸乙酯提取物。
4.根据权利要求3所述的应用，其特征在于，步骤①中，加5倍量75％乙醇浸泡12h。
5.根据权利要求3所述的应用，其特征在于，步骤②中，水浴加热回流提取2次，每次2h。
6.根据权利要求3所述的应用，其特征在于，步骤④中，振摇萃取5次。
7.根据权利要求3所述的应用，其特征在于，步骤⑥中，用温度为50℃的热水重新溶解浸膏。
8.根据权利要求2所述的应用，其特征在于，所述宽叶苔草乙酸乙酯提取物的作用浓度范围为：0.01-1mg/ml。
9.根据权利要求8所述的应用，其特征在于，所述宽叶苔草乙酸乙酯提取物的浓度为：0.825mg/ml。
10.根据权利要求1所述的应用，其特征在于，所述产品包括：食品、药品、保健品和化妆品。
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