CN111387505B - 一种对黑色素合成抑制的生菜提取物及生菜配套栽培方法 - Google Patents
一种对黑色素合成抑制的生菜提取物及生菜配套栽培方法 Download PDFInfo
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Abstract
本发明一方面公开了一种生菜的栽培方法,即选取品种为“Lollo Rossa”的生菜种子,在育苗穴盘中培养10天,定植于温室中的漂浮板栽培30天后冷冻保存;另一方面本发明还公开了这种生菜具有黑色素抑制作用的提取物的制备方法,即将生菜新鲜样品通过研磨、提取、离心和真空冷冻干燥,最后溶解于含有0.1%二甲基亚砜(DMSO)的灭菌超纯水中;再一方面,本发明公开这种提取物在制备祛斑化妆品、药品、保健品、日化用品或功能性原料中的应用。本发明提供的这种生菜提取物,来自天然植物,没有细胞毒性,同时又显著抑制黑色素的合成,有着广泛的用途。
Description
技术领域
本发明涉及植物生理领域,尤其涉及一种生菜配套栽培方法及对黑色素合成抑制的生菜提取物的制备和应用。
背景技术
皮肤肤色的深浅主要由皮肤黑色素含量及分布情况决定,黑色素能减少紫外线对皮肤的伤害。然而,当过量黑色素异常积累并沉着过度时,会引起老年斑、黑斑病、雀斑等严重影响皮肤美观的疾病。因此,具有美白功效的祛斑化妆品及其功能性原料的研究与开发日趋活跃。
黑色素主要包括两种高聚合物醌类物质,分别是真黑素和褐黑素。黑色素生物合成步骤主要包括:酪氨酸酶催化酪氨酸形成多巴醌;多巴醌氧化生成多巴和多巴色素,其中的多巴被催化后再次生成多巴醌;多巴色素的反应产物5,6-二羟基吲哚和5,6-二羟基吲哚羧酸经氧化生成真黑素;在半胱氨酸或者谷胱甘肽存在的条件下,多巴醌会转变成半胱氨酰多巴,最后生成褐黑素。此代谢过程中关键底物是酪氨酸,限速酶是酪氨酸酶,氧气参与了代谢。抑制黑色素合成的机制主要是通过抑制酪氨酸酶活性、抑制多巴色素异构酶活性、抑制氧化反应等。检索文献和专利,未见生菜抑制黑色素合成的报道。
目前市场上主要的抑制黑色素合成的美白成分有熊果苷、曲酸衍生物、抗坏血酸衍生物、氢醌衍生物等,这些美白剂有显著的美白效果,但也存在皮肤刺激、光敏感、细胞毒性和容易变色等问题。
因此,本领域的技术人员致力于开发一种来源于天然植物、没有细胞毒性的、安全的抑制黑色素合成的物质,可用于皮肤美白功效,比如祛斑化妆品及其功能性原料。
发明内容
有鉴于现有技术的上述缺陷,本发明所要解决的技术问题是开发一种来源于天然植物、没有细胞毒性的、安全的抑制黑色素合成的物质。
为实现上述目的,本发明一方面提供了一种生菜的的栽培方法,具体步骤包括:
步骤a、育苗,选取生菜种子,在育苗穴盘中培养10天,获得生菜的苗;
步骤b、栽培,然后将步骤a获得的苗定植于温室中漂浮在培养液之上的漂浮板,栽培持续30天,得到生菜;
步骤c、冷冻保存生菜。
进一步地,步骤a中生菜为生菜(Lactuca sativa L.),品种为“Lollo Rossa”。
进一步地,步骤b中,培养液中设置有4.5mmol/L~18mmol/L的甘氨酸。
进一步地,步骤b中,栽培的温度为:日间平均温度为22℃,夜间平均温度为16℃。
进一步地,步骤c的具体步骤为:将步骤b获得的生菜的全株经液氮速冻,然后放入-80℃冰箱保存。
另一方面,本发明公开了一种生菜提取物提取方法,包括以下步骤:
步骤1)、取0.2g新鲜的生菜的样品于液氮中研磨成粉,置于1.5mL离心管中,加入1mL甲醇水提取液,涡旋混匀后置于室温下超声提取30min,获得生菜的超声提取液;
步骤2)、将步骤1)获得的超声提取液移入4℃冰箱,冷藏提取12h,获得生菜的冷藏提取液;
步骤3)、将步骤2)获得的冷藏提取液在转速为10000r/min的离心机中离心10min,取上清液0.5mL真空冷冻干燥,并复溶于含有0.1%二甲基亚砜(DMSO)的200μL灭菌超纯水中,获得生菜提取物。
进一步地,步骤1)中,甲醇水提取液中甲醇和水的体积比为4:1。
进一步地,步骤3)中,生菜提取物的最终浓度为0.5mg/μL。
进一步地,生菜提取物能够抑制动物体黑色素合成。
再一方面,本发明公开了根据以上提取方法制备的生菜提取物在制备祛斑化妆品、药品、保健品、日化用品或功能性原料中的应用。
与现有技术相比,本发明具有的有益效果:
1、本发明通过黑色素合成抑制的体外实验和动物实验证实该生菜提取物具有黑色素合成抑制的显著作用,有美白皮肤的作用,可被广泛应用,比如应用于护肤品或化妆品,特别是作为美白功效的祛斑化妆品及其功能性原料;也可用作药品和保健品等用途。
2、本发明通过对B16小鼠黑色素瘤细胞系的细胞毒性实验证实该生菜提取物无细胞毒性,是安全的抑制黑色素合成的物质,且来源于天然植物。
3、通过对斑马鱼胚胎的动物实验证实该生菜提取物对斑马鱼胚胎发育没有影响,是安全的抑制黑色素合成的物质。
4、本发明提供的该生菜的栽培方法和生菜提取物的提取方法,简单实用,便于推广。
以下将结合附图对本发明的构思、具体结构及产生的技术效果作进一步说明,以充分地了解本发明的目的、特征和效果。
附图说明
图1为本发明生菜提取物对B16小鼠黑色素瘤细胞黑色素含量的抑制作用试验结果示意图;
图2为本发明生菜提取物对斑马鱼胚胎黑色素合成的抑制作用示意图;
图3为图2的部分区域局部放大示意图。
具体实施方式
以下参考说明书附图介绍本发明的优选实施例,使其技术内容更加清楚和便于理解。本发明可以通过许多不同形式的实施例来得以体现,本发明的保护范围并非仅限于文中提到的实施例。
实施例1
1、生菜栽培
a、育苗,选取品种“Lollo Rossa”的生菜(Lactuca sativa L.)种子,在育苗穴盘中培养10天,得到生菜苗;
b、栽培,将生菜苗定植于温室中漂浮在培养液之上的漂浮板,栽培持续30天,得到生菜栽培30天,栽培期间,培养液分组分别设置为含有4.5mmol/L甘氨酸的S13组、含有9mmol/L甘氨酸的S15组、含有18mmol/L甘氨酸的S17组,栽培期间温度控制为:日间平均温度为22℃,夜间均温为16℃,收获生菜;
c、将生菜的全株经液氮速冻,然后放入-80℃冰箱保存。
2、提取物制备
步骤1)、分别取三个新鲜的生菜样品0.2g于液氮中研磨成粉,置于1.5mL离心管(EP管)中,加入1mL甲醇水提取液,其中甲醇和水的体积比为4:1;涡旋混匀后置于室温下超声提取30min,得到生菜超声提取液;
步骤2)、随后将超声提取液移入4℃冰箱,冷藏提取12h,获得生菜冷藏提取液;
步骤3)、将生菜冷藏提取液在转速为10000r/min的离心机内离心10min,取上清液0.5mL真空冷冻干燥,并复溶于含有0.1%二甲基亚砜(DMSO)的200μL灭菌超纯水中,得到生菜提取物,生菜提取物的最终浓度为0.5mg/μL。
实施例2
提取物效果监测试验:
1、测定生菜提取物对B16小鼠黑色素瘤细胞系的细胞毒性
步骤1)、将B16小鼠黑色素瘤细胞用培养基重悬后铺96孔板,每孔约5000个细胞,37℃,5%的CO2培养24h后弃液;
步骤2)、用含有多种氨基酸和葡萄糖的培养基(DMEM培养基)将三个上述生菜提取液稀释,即分别将10μL生菜提取物(S13组、S15组、S17组)和90μL DMEM培养基混合加入细胞中;以没有加生菜提取物的B16小鼠黑色素瘤细胞为对照;对于加生菜提取物的细胞样品和对照细胞样品,每个样品都设置5个复孔;
步骤3)、48小时后,检测生菜提取物对细胞生长的抑制效率。
结果显示,与对照相比,生菜提取物S13组、S15组、S17组对B16小鼠黑色素瘤细胞没有毒性和也没有增殖抑制作用。
2、测定生菜提取物抑制黑色素合成效果的体外试验
步骤1)、对于上述B16小鼠黑色素瘤细胞培养液,包括三种加生菜提取物的细胞样品的S13组、S15组、S17组和对照细胞样品的对照组(Mock组),在离心机上离心,弃上清,得到沉淀;用磷酸缓冲盐溶液(PBS)洗沉淀2次,离心,弃上清,在沉淀中加入200μL纯水重悬,随后加入1mL乙醇/乙醚混合液(乙醇与乙醚的体积比为1:1),摇匀后静置15分钟,随后低速离心,弃上清,得到细胞沉淀。
步骤2)、向所得细胞沉淀加入1mL 10%的DMSO(以1mol/L NaOH配制)溶液,振荡混合后于80℃封口,放置30分钟,得到的溶液含有黑色素,为黑色素总量待测液。
步骤3)、将上述黑色素总量待测液分别使用分光光度计测定波长为470nm的吸光值。
各组的黑色素总量如图1所示,示意图说明与对照相比,生菜提取物S13组、S15组和S17组都对黑色素形成均具有抑制作用,其中S13组效果最佳。实验结果证明:本发明提供的生菜提取物能显著抑制B16小鼠黑色素瘤细胞内合成的黑色素总量。
2、测定生菜提取物抑制黑色素含量效果的动物试验
生菜提取物对斑马鱼表皮黑色素合成的抑制效果:将受精后0.75h的斑马鱼胚胎分别暴露于S13组、S15组、S17组三个不同生菜提取物中,48小时后观察斑马鱼胚胎结果。
实验结果如图2,图3所示,三个提取物试验组的胚胎发育程度与对照的Mock组的相当,说明生菜提取物S13组、S15组和S17组较对照的Mock组来说,都不影响胚胎生长发育;从各组中斑马鱼胚胎的体表颜色来看,生菜提取物S13组、S15组和S17组较对照Mock组颜色浅,其中S13组图片中斑马鱼胚胎的体表颜色最浅,说明生菜提取物S13组、S15组和S17组较对照Mock组来说,对黑色素形成均具有抑制作用,其中S13组效果最佳,从而得出结论:这种生菜提取物能有效抑制斑马鱼胚胎黑色素合成,同时不会影响斑马鱼胚胎发育。
以上详细描述了本发明的较佳具体实施例。应当理解,本领域的普通技术无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此,凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案,皆应在由权利要求书所确定的保护范围内。
Claims (6)
1.一种作为对黑色素合成抑制提取物原料的生菜的栽培方法,其特征在于,所述栽培方法包括以下步骤:
步骤a、育苗,选取所述生菜种子,在育苗穴盘中培养10天,获得所述生菜的苗;
步骤b、栽培,然后将步骤a获得的所述苗定植于温室中漂浮在培养液之上的漂浮板,所述栽培持续30天,得到所述生菜;
步骤c、冷冻保存所述生菜;所述步骤a中,所述生菜的品种为‘Lollo Rossa’;所述步骤b中,所述培养液中设置有4.5mmol/L~18mmol/L的甘氨酸。
2.如权利要求1所述的一种生菜的栽培方法,其特征在于,步骤b中,所述栽培的温度为:日间平均温度为22℃,夜间平均温度为16℃。
3.如权利要求1所述的一种生菜的栽培方法,其特征在于,步骤c的具体步骤为:将步骤b获得的所述生菜的全株经液氮速冻,然后放入-80℃冰箱保存。
4.一种利用权利要求1所述的栽培方法得到的所述生菜的生菜提取物提取方法,其特征在于,包括以下步骤:
步骤⑴、取0.2 g新鲜的所述生菜的样品于液氮中研磨成粉,置于1.5 mL离心管中,加入1 mL甲醇水提取液,涡旋混匀后置于室温下超声提取30 min,获得所述生菜的超声提取液;
步骤⑵、将步骤⑴获得的所述超声提取液移入4℃冰箱,冷藏提取12 h,获得所述生菜的冷藏提取液;
步骤⑶、将步骤⑵获得的所述冷藏提取液在转速为10000 r/min的离心机中离心10min,取上清液0.5 mL真空冷冻干燥,并复溶于含有0.1%二甲基亚砜(DMSO)的200 μL灭菌超纯水中,获得所述生菜提取物;
所述步骤⑴中,所述甲醇水提取液中甲醇和水的体积比为4:1;所述步骤⑶中,所述生菜提取物的最终浓度为0.5 mg /μL。
5.根据权利要求4的所述提取方法制备的所述生菜提取物,其特征在于,所述生菜提取物能够抑制动物体黑色素合成。
6.根据权利要求4的所述提取方法制备的所述生菜提取物在制备保健品或日化用品中的应用。
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