CN108420074A - 一种酪氨酸酶活性抑制剂及其应用 - Google Patents
一种酪氨酸酶活性抑制剂及其应用 Download PDFInfo
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Abstract
本发明属于生物领域,具体涉及生物酶,涉及一种酪氨酸酶活性的抑制剂,以及该抑制剂在抑制酪氨酸酶活性方面的应用。本发明所提供的具有酪氨酸酶抑制活性的化合物可应用于制备抗果蔬酶促褐变抑制剂、化妆品美白剂以及其他需要抑制酪氨酸酶活性的病症的药物中,可以作为活性成分,不加或加入食品添加剂辅料、化妆品辅料或药剂上接受的辅料,按照相应剂型的制备方法制成食品或保健品,也可以用于果蔬保鲜液。
Description
技术领域:
本发明属于生物领域,涉及酪氨酸酶酶活性的控制,具体涉及一种酪氨酸酶活性抑制剂及其应用。
背景技术:
酪氨酸酶(Tyrosinase,EC 1.14.18.1)是一种75kD含铜酶,来源于胚胎神经鞘细胞,是黑色素代谢和儿茶酚胺的关键酶。Spritz等人证实TYR是一种铜结合蛋白,有铜A和铜B位点。离子铜特异性能与人TYR结合,一个位点的铜结合可以促进另一个位点的铜结合。组氨酸在铜B位点协调其结合。酪氨酸酶是生物体黑色素合成的关键酶,兼有加氧酶和氧化酶的双重功能,它能够把单酚催化成二酚,并把二酚氧化成醌;醌在非酶促条件下形成最终的反应产物黑色素。酪氨酸酶抑制剂就是通过抑制酪氨酸酶活性从而抑制黑色素生成。对于人体健康而言,酪氨酸酶的异常过量表达可导致人体的色素沉着性疾病;对于果树加工而言,由酪氨酸酶引起的酶促褐变不仅影响产品的色泽、外观,而且会破坏必需产品的氨基酸、降低其营养和消化吸收,并最终严重影响产品的营养价值和市场价值。在医药领域,酪氨酸酶抑制剂就是通过抑制酪氨酸酶活性从而抑制黑色素生成,因此酪氨酸酶抑制剂通过破坏酪氨酸酶活性,从而达到减少皮肤中黑色素的目的,进而用于治疗目前常见的色素沉着疾病如雀斑、黄褐斑、老年斑和黑色素瘤等,可广泛应用于医药和化妆品行业。在果蔬保藏领域,通过抑制酪氨酸酶的活性可以有效地减少果蔬褐变,提高果蔬的保藏品质。
辣木为辣木科辣木属植物,原产于印度北部喜马拉雅区域,辣木不仅可以食用,在热带、亚热带地区还用作植物药材而备受重视。近年来,辣木在我国广东、云南和海南等 都有引种栽培,其产品的开发成为热点,现有研究表明,长期服用辣木籽可以有效地起到抗衰老的作用。然而,尚未有研究将辣木叶提取物用于抑制酪氨酸酶的活性。
发明内容:
基于现有技术发展进程的缓慢和不足,本发明旨在提供一种酪氨酸酶活性的抑制剂,以及该抑制剂在抑制酪氨酸酶活性方面的应用,本发明采用天然的辣木叶为原料,经自然友好的方法提取获得了具有良好的酪氨酸酶抑制活性的抑制剂,以及该抑制剂在抑制酪氨酸酶活性中的应用。
为了解决以上技术问题,本发明采用如下技术手段:
本发明首先请求保护辣木叶提取物在抑制酪氨酸酶活性中的应用。
本发明还涉及一种酪氨酸酶活性抑制剂,所述酪氨酸酶活性抑制剂是以辣木叶为原料经提取制备而成,所述酪氨酸酶活性抑制剂经由以下步骤制备而成:步骤一,称取适量的干燥辣木叶,将其粉碎至250-300目,得到辣木叶粉末;步骤二,将粉碎好的辣木叶粉末与水按照质量比3:7-8混合,搅拌均匀,而后加入辣木叶粉末质量0.2-0.3倍量的氢氧化钠,搅拌混匀后,将混合物在50-55℃下加热20-25min,得到碱处理的混合物;步骤三,向步骤二制得的碱处理的混合物加入盐酸溶液,调节pH至6.8-7.2,而后进行离心分离,12000rpm离心处理10min,分别得到上清液和沉淀,收集上清液;步骤四,向步骤三得到的沉淀干燥,用15倍质量的乙醇于65-68℃下回流提取2次,每次3小时,收集并合并提取液;步骤五,将步骤四制得的提取液与步骤三离心分离得到的上清液混合,真空冷冻干燥,即得到所述的酪氨酸酶活性抑制剂。
优选地,所述酪氨酸酶活性抑制剂经由以下步骤制备而成:步骤一,称取适量的干燥辣木叶,将其粉碎至280目,得到辣木叶粉末;步骤二,将粉碎好的辣木叶粉末与水按照质量比3:7混合,搅拌均匀,而后加入辣木叶粉末质量0.25倍量的氢氧化钠,搅拌混匀后,将混合物在53℃下加热22min,得到碱处理的混合物;步骤三,向步骤二制得的碱处理的混合物加入盐酸溶液,调节pH至7.0,而后进行离心分离,12000rpm离心处理10min,分别得到上清液和沉淀,收集上清液;步骤四,向步骤三得到的沉淀干燥,用15倍质量的乙醇于66℃下回流提取2次,每次3小时,收集并合并提取液;步骤五,将步骤四制得的提取液与步骤三离心分离得到的上清液混合,真空冷冻干燥,即得到所述的酪氨酸酶活性抑制剂。
优选地,所述酪氨酸酶活性抑制剂经由以下步骤制备而成:步骤一,称取适量的干燥辣木叶,将其粉碎至250目,得到辣木叶粉末;步骤二,将粉碎好的辣木叶粉末与水按照质量比3:7混合,搅拌均匀,而后加入辣木叶粉末质量0.2倍量的氢氧化钠,搅拌混匀后,将混合物在55℃下加热20min,得到碱处理的混合物;步骤三,向步骤二制得的碱处理的混合物加入盐酸溶液,调节pH至6.8,而后进行离心分离,12000rpm离心处理10min,分别得到上清液和沉淀,收集上清液;步骤四,向步骤三得到的沉淀干燥,用15倍质量的乙醇于65℃下回流提取2次,每次3小时,收集并合并提取液;步骤五,将步骤四制得的提取液与步骤三离心分离得到的上清液混合,真空冷冻干燥,即得到所述的酪氨酸酶活性抑制剂。
优选地,所述酪氨酸酶活性抑制剂经由以下步骤制备而成:步骤一,称取适量的干燥辣木叶,将其粉碎至300目,得到辣木叶粉末;步骤二,将粉碎好的辣木叶粉末与水按照质量比3: 8混合,搅拌均匀,而后加入辣木叶粉末质量0.3倍量的氢氧化钠,搅拌混匀后,将混合物在50℃下加热25min,得到碱处理的混合物;步骤三,向步骤二制得的碱处理的混合物加入盐酸溶液,调节pH至7.2,而后进行离心分离,12000rpm离心处理10min,分别得到上清液和沉淀,收集上清液;步骤四,向步骤三得到的沉淀干燥,用15倍质量的乙醇于68℃下回流提取2次,每次3小时,收集并合并提取液;步骤五,将步骤四制得的提取液与步骤三离心分离得到的上清液混合,真空冷冻干燥,即得到所述的酪氨酸酶活性抑制剂。
本发明还请求保护所述的酪氨酸酶活性抑制剂在制备用于抑制酪氨酸酶活性的药物、化妆品、保鲜剂或食品中的用途,将其作为有效成分可以有效地抑制酪氨酸酶的活性。
基于以上技术方案,本发明具有如下有益效果:
首先,在探究辣木叶应用的过程中,申请人偶然发现了辣木叶提取物具有抑制水果褐变的作用,进而研究发现基于辣木叶的提取物对于水果褐变过程中关键的酪氨酸酶类具有显著的抑制作用,进而提出了将其作为酪氨酸酶抑制剂的运用,其中辣木叶提取物的上述应用是申请人偶然所得,在现有技术中尚无报道,弥补了辣木叶提取物运用的空白。
其次,本发明以天然原料-辣木叶为原料,经过碱处理以及醇提工艺等制备得到辣木叶提取物,其加工的原料为天然的辣木叶原料,其加工中也仅涉及氢氧化钠、盐酸以及乙醇等三种常规试剂的使用,且氢氧化钠和盐酸在加工过程中已经中和、乙醇在后续的加工中也已经蒸发,因而,实际所制备的辣木叶提取物中并不包含任何对人体有害的化学试剂,其符合天然、健康的生态发展理念,并且经动物经口试验证明,本发明的辣木叶提取物对动物是安全无毒的。
再次,本发明的辣木叶提取物对于酪氨酸酶具有非常好的抑制作用,其对于酪氨酸酶的抑制率可以高达78%以上,高于常用的熊果苷和曲酸对于酪氨酸酶的抑制率。
本发明所提供的具有酪氨酸酶抑制活性的化合物可应用于制备抗果蔬酶促褐变抑制剂、化妆品美白剂以及其他需要抑制酪氨酸酶活性的病症的药物中,可以作为活性成分,不加或加入食品添加剂辅料、化妆品辅料或药剂上接受的辅料,按照相应剂型的制备方法制成食品或保健品,也可以用于果蔬保鲜液。
附图说明:
图1:将本发明的酪氨酸酶抑制剂用于梨的保鲜试验,其中左侧为喷涂本发明酪氨酸抑制剂的试验样品经贮藏150天后的结果,右侧为喷涂市售的梨用保鲜剂的试验样品经贮藏150天后的结果。
具体实施方式:
实施例1:一种酪氨酸酶活性抑制剂,所述酪氨酸酶活性抑制剂经由以下步骤制备而成:步骤一,称取适量的干燥辣木叶,将其粉碎至280目,得到辣木叶粉末;步骤二,将粉碎好的辣木叶粉末与水按照质量比3:7混合,搅拌均匀,而后加入辣木叶粉末质量0.25倍量的氢氧化钠,搅拌混匀后,将混合物在53℃下加热22min,得到碱处理的混合物;步骤三,向步骤二制得的碱处理的混合物加入盐酸溶液,调节pH至7.0,而后进行离心分离,12000rpm离心处理10min,分别得到上清液和沉淀,收集上清液;步骤四,向步骤三得到的沉淀干燥,用15倍质量的乙醇于66℃下回流提取2次,每次3小时,收集并合并提取液;步骤五,将步骤四制得的提取液与步骤三离心分离得到的上清液混合,真空冷冻干燥,即得到所述的酪氨酸酶活性抑制剂。
实施例2:一种酪氨酸酶活性抑制剂,所述酪氨酸酶活性抑制剂经由以下步骤制备而成:步骤一,称取适量的干燥辣木叶,将其粉碎至250目,得到辣木叶粉末;步骤二,将粉碎好的辣木叶粉末与水按照质量比3:7混合,搅拌均匀,而后加入辣木叶粉末质量0.2倍量的氢氧化钠,搅拌混匀后,将混合物在55℃下加热20min,得到碱处理的混合物;步骤三,向步骤二制得的碱处理的混合物加入盐酸溶液,调节pH至6.8,而后进行离心分离,12000rpm离心处理10min,分别得到上清液和沉淀,收集上清液;步骤四,向步骤三得到的沉淀干燥,用15倍质量的乙醇于65℃下回流提取2次,每次3小时,收集并合并提取液;步骤五,将步骤四制得的提取液与步骤三离心分离得到的上清液混合,真空冷冻干燥,即得到所述的酪氨酸酶活性抑制剂。
实施例3:一种酪氨酸酶活性抑制剂,所述酪氨酸酶活性抑制剂经由以下步骤制备而成:步骤一,称取适量的干燥辣木叶,将其粉碎至300目,得到辣木叶粉末;步骤二,将粉碎好的辣木叶粉末与水按照质量比3: 8混合,搅拌均匀,而后加入辣木叶粉末质量0.3倍量的氢氧化钠,搅拌混匀后,将混合物在50℃下加热25min,得到碱处理的混合物;步骤三,向步骤二制得的碱处理的混合物加入盐酸溶液,调节pH至7.2,而后进行离心分离,12000rpm离心处理10min,分别得到上清液和沉淀,收集上清液;步骤四,向步骤三得到的沉淀干燥,用15倍质量的乙醇于68℃下回流提取2次,每次3小时,收集并合并提取液;步骤五,将步骤四制得的提取液与步骤三离心分离得到的上清液混合,真空冷冻干燥,即得到所述的酪氨酸酶活性抑制剂。
实施例4:酪氨酸酶活性抑制试验(本试验委托北京市亦庄生物技术产业园区某资质企业进行,参照发明专利CN201410017256 实施例2的方法)
实验材料:酪氨酸酶、熊果苷、曲酸、L-DOPA。
受试药物:实施例1-3的酪氨酸酶活性抑制剂,分别溶解于DMSO中,制备得25mg/mL母液;熊果苷、曲酸溶解于DMSO中,制得25mg/mL母液。
实验方法:96孔板中每孔加入30μL 25μg/mL酪氨酸酶及30μL待测样品,混匀,25℃孵育10min后,每孔加入50μL 5mM L-DOPA启动反应,25℃避光孵育5min,于475nm处读取吸光度值(每孔均做空白对照<以缓冲液代替酪氨酸酶>以扣除提取物颜色对吸光度值的影响)。
实验结果如表1所示。
表1 酪氨酸酶活性抑制试验
由以上试验可知,本发明实施例1-3的酪氨酸酶活性抑制剂对于酪氨酸酶的活性具有良好的抑制作用,其对于酪氨酸酶活的抑制率高达78.2%以上,高于常用的熊果苷和曲酸对于酪氨酸酶的抑制活性。
实施例5:动物经口毒性试验(本试验委托北京市亦庄生物技术产业园区某资质企业进行)
小鼠急性毒性试验:采用本发明实施例1-3的酪氨酸酶活性抑制剂加水制成5mg/mL的溶液,替代小鼠饲养用水,连续观察28天,试验期间未见死亡和异常毒性反应,试验结束,解剖试验小鼠,观察肝、肾、脾、胃、心、肺和肠,均未见异常改变。按急性毒性标准,该样品属于无毒级。
小鼠慢性毒性试验:采用本发明实施例1-3的酪氨酸酶活性抑制剂加水制成0.25mg/mL的溶液,替代小鼠饲养用水,连续观察120天,结果显示:饮用本发明实施例1-3的产品水溶液的小鼠行为活动正常,皮毛光滑,体重正常增加,摄食饮水正常,血液学和血液生化指标均在正常范围内波动,骨髓和尿常规指标均在正常范围内波动,组织脏器未见异常,整体而言未见毒理反应。
由以上实验可知,本发明的产品是安全、无毒的。
实施例6:果蔬保鲜试验
将本发明实施例1的酪氨酸酶抑制剂加水制成0.3mg/ml浓度的溶液,购买市售的梨用保鲜喷雾,在待保藏的梨表面均匀喷洒本发明实施例1的酪氨酸酶抑制剂溶液或者喷洒市售的梨用保鲜喷雾,而后按照标准贮藏程序进行气调贮藏150天后,图1左侧为喷洒本发明实施例1的酪氨酸酶抑制剂的试验样品经贮藏150天后的结果,右侧为喷涂市售的梨用保鲜剂的试验样品经贮藏150天后的结果。由图1的结果可知,本发明的酪氨酸酶抑制剂可以有效的防止梨的果皮褐变。
Claims (7)
1.一种酪氨酸酶活性抑制剂,所述酪氨酸酶活性抑制剂是以辣木叶为原料经提取制备而成,所述酪氨酸酶活性抑制剂经由以下步骤制备而成:步骤一,称取适量的干燥辣木叶,将其粉碎至250-300目,得到辣木叶粉末;步骤二,将粉碎好的辣木叶粉末与水按照质量比3:7-8混合,搅拌均匀,而后加入辣木叶粉末质量0.2-0.3倍量的氢氧化钠,搅拌混匀后,将混合物在50-55℃下加热20-25min,得到碱处理的混合物;步骤三,向步骤二制得的碱处理的混合物加入盐酸溶液,调节pH至6.8-7.2,而后进行离心分离,12000rpm离心处理10min,分别得到上清液和沉淀,收集上清液;步骤四,向步骤三得到的沉淀干燥,用15倍质量的乙醇于65-68℃下回流提取2次,每次3小时,收集并合并提取液;步骤五,将步骤四制得的提取液与步骤三离心分离得到的上清液混合,真空冷冻干燥,即得到所述的酪氨酸酶活性抑制剂。
2.根据权利要求1所述的酪氨酸酶活性抑制剂,其特征在于,所述酪氨酸酶活性抑制剂经由以下步骤制备而成:步骤一,称取适量的干燥辣木叶,将其粉碎至280目,得到辣木叶粉末;步骤二,将粉碎好的辣木叶粉末与水按照质量比3:7混合,搅拌均匀,而后加入辣木叶粉末质量0.25倍量的氢氧化钠,搅拌混匀后,将混合物在53℃下加热22min,得到碱处理的混合物;步骤三,向步骤二制得的碱处理的混合物加入盐酸溶液,调节pH至7.0,而后进行离心分离,12000rpm离心处理10min,分别得到上清液和沉淀,收集上清液;步骤四,向步骤三得到的沉淀干燥,用15倍质量的乙醇于66℃下回流提取2次,每次3小时,收集并合并提取液;步骤五,将步骤四制得的提取液与步骤三离心分离得到的上清液混合,真空冷冻干燥,即得到所述的酪氨酸酶活性抑制剂。
3.根据权利要求1所述的酪氨酸酶活性抑制剂,其特征在于,所述酪氨酸酶活性抑制剂经由以下步骤制备而成:步骤一,称取适量的干燥辣木叶,将其粉碎至250目,得到辣木叶粉末;步骤二,将粉碎好的辣木叶粉末与水按照质量比3:7混合,搅拌均匀,而后加入辣木叶粉末质量0.2倍量的氢氧化钠,搅拌混匀后,将混合物在55℃下加热20min,得到碱处理的混合物;步骤三,向步骤二制得的碱处理的混合物加入盐酸溶液,调节pH至6.8,而后进行离心分离,12000rpm离心处理10min,分别得到上清液和沉淀,收集上清液;步骤四,向步骤三得到的沉淀干燥,用15倍质量的乙醇于65℃下回流提取2次,每次3小时,收集并合并提取液;步骤五,将步骤四制得的提取液与步骤三离心分离得到的上清液混合,真空冷冻干燥,即得到所述的酪氨酸酶活性抑制剂。
4.根据权利要求1所述的酪氨酸酶活性抑制剂,其特征在于,所述酪氨酸酶活性抑制剂经由以下步骤制备而成:步骤一,称取适量的干燥辣木叶,将其粉碎至300目,得到辣木叶粉末;步骤二,将粉碎好的辣木叶粉末与水按照质量比3: 8混合,搅拌均匀,而后加入辣木叶粉末质量0.3倍量的氢氧化钠,搅拌混匀后,将混合物在50℃下加热25min,得到碱处理的混合物;步骤三,向步骤二制得的碱处理的混合物加入盐酸溶液,调节pH至7.2,而后进行离心分离,12000rpm离心处理10min,分别得到上清液和沉淀,收集上清液;步骤四,向步骤三得到的沉淀干燥,用15倍质量的乙醇于68℃下回流提取2次,每次3小时,收集并合并提取液;步骤五,将步骤四制得的提取液与步骤三离心分离得到的上清液混合,真空冷冻干燥,即得到所述的酪氨酸酶活性抑制剂。
5.辣木叶提取物在抑制酪氨酸酶活性中的应用。
6.根据权利要求5所述的辣木叶提取物在抑制酪氨酸酶活性中的应用,其特征在于,所述辣木叶提取物采用如下方法制备得到:步骤一,称取适量的干燥辣木叶,将其粉碎至250-300目,得到辣木叶粉末;步骤二,将粉碎好的辣木叶粉末与水按照质量比3:7-8混合,搅拌均匀,而后加入辣木叶粉末质量0.2-0.3倍量的氢氧化钠,搅拌混匀后,将混合物在50-55℃下加热20-25min,得到碱处理的混合物;步骤三,向步骤二制得的碱处理的混合物加入盐酸溶液,调节pH至6.8-7.2,而后进行离心分离,12000rpm离心处理10min,分别得到上清液和沉淀,收集上清液;步骤四,向步骤三得到的沉淀干燥,用15倍质量的乙醇于65-68℃下回流提取2次,每次3小时,收集并合并提取液;步骤五,将步骤四制得的提取液与步骤三离心分离得到的上清液混合,真空冷冻干燥,即得到所述的辣木叶提取物。
7.权利要求1-4任一项所述的酪氨酸酶活性抑制剂在制备用于抑制酪氨酸酶活性的药物、化妆品、保鲜剂或食品中的用途,将其作为有效成分可以有效地抑制酪氨酸酶的活性。
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