CN114469814A - 一种睡莲花提取物及其制备方法和应用 - Google Patents
一种睡莲花提取物及其制备方法和应用 Download PDFInfo
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- CN114469814A CN114469814A CN202210161591.5A CN202210161591A CN114469814A CN 114469814 A CN114469814 A CN 114469814A CN 202210161591 A CN202210161591 A CN 202210161591A CN 114469814 A CN114469814 A CN 114469814A
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- water lily
- lily flower
- nymphaea tetragona
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Abstract
本发明公开了一种睡莲花提取物及其制备方法和应用,属于植物提取技术领域。所述制备方法包括:(1)将睡莲花干制品按照1g:20mL~60mL的料液比加入到体积比为50%~90%的有机溶剂水溶液中,于20~65℃条件下浸提,分离得到萃取液;(2)调节萃取液的pH值至7~9,然后去除有机溶剂,干燥后制得睡莲花提取物。通过本发明技术实现了睡莲花中护肤功效成分的高效富集提取,本发明提供的提取物中主要成分鞣花酸和异槲皮苷含量相对高。该提取物具有较强的抗氧化和抗皱能力,在化妆品、食品、医药产品等领域中具有极大的应用前景。
Description
技术领域
本发明涉及植物提取技术领域,具体涉及一种睡莲花提取物及其制备方法和应用。
背景技术
睡莲(Nymphaea tetragona Georgi.)是睡莲属(Nymphaea)多年生水生草本植物,广泛分布于温带及热带地区。睡莲花大形,浮在或高于水面。花瓣呈白色、黄色、粉色或蓝色等。该属植物多生长在池沼地,也是睡莲科中分布最广的属。目前在全世界睡莲约有35种,睡莲在国内各省区均有栽培,花主供观赏,大部分随着花期的结束而凋谢腐烂成为池塘养料废弃物。值得一提的是,睡莲花具有悠久的食用历史。研究表明,睡莲花除了含有碳水化合物、脂肪、蛋白质等常规的营养成分外,还含有黄酮、酚酸、生物碱、木脂素及多糖等多种活性成分,具有抗氧化、抗菌、抗炎、抗辐射、降血糖和降血压等多种生物活性,表明睡莲花具有一定的食用和药用价值。
迄今为止,人们对睡莲花成分与活性的研究已经取得了一定的进展。如研究发现,睡莲花茶中含有抗氧化和抗癌活性强的槲皮素和查耳酮等化合物。徐辉等初步测定了香水莲花的基本化学成分,纯化并鉴定了甲酯化半乳糖醛酸、β-谷甾醇和没食子酸等活性成分,并证明其具有降血脂的功效(香水莲花的化学成分及活性功能研究[D].南京农业大学,2008.83)。任红荣等测定了香水莲花的多酚类组分,并进行了抑制酪氨酸酶活性研究(香水莲花醇提取物对酪氨酸酶抑制作用的研究[J].日用化学品科学.2009,第012期)。袁茹玉利用HPLC-DAD和HPLC-ESI(+/-)-MS2对不同睡莲品种的茶汤进行分析,检测到茶汤中含有16种类黄酮物质。然而,在实际睡莲花资源开发利用方面,其主要加工工艺仍为干制,仅少部分加工成花茶。对睡莲花提取利用往往是获得粗水提物,未明确睡莲花相应活性的实际功效成分。
皮肤老化是由环境因素和生活习惯因素而导致的老化,主要表现为皱纹增多、皮肤松弛、粗糙等。常见的皮肤老化包括自然老化和光老化两种。后者指的是由于环境因素,尤其是UV辐射而引起的皮肤老化。UV辐射是加速皮肤老化进程的主要因素。引起皮肤老化的机制复杂,主要涉及氧化应激、DNA损伤、胶原蛋白损失、炎症反应等方面。人们对应对皮肤老化尤其是抗皱相关护肤品的需求与日俱增,同时考虑到安全性,人们也越来越重视天然产物在护肤品中的应用,各种植物提取物(尤其是来自花部位)作为功效成分的护肤品成为市场的热点。
目前针对睡莲花护肤功效的报道仅是体外美白相关酶活性的研究,相关产品的研制和开发未见报道。而睡莲花提取物在抗皮肤老化方面的功效也未见报道。
发明内容
本发明的目的在于提供一种针对睡莲花的活性成分进行高效提取的方法,并对睡莲花提取物的护肤功效进行深入研究,开发其新的功能,以填补目前睡莲花提取物在抗皱等护肤功效和功效成分的研究空缺。
为实现上述目的,本发明采用如下技术方案:
一种睡莲花提取物的制备方法,包括以下步骤:
(1)将睡莲花干制品按照1g:20mL~60mL的料液比加入到体积比为50%~90%的有机溶剂水溶液中,于20~65℃条件下浸提,分离得到萃取液;
(2)调节萃取液的pH值至7~9,然后去除有机溶剂,干燥后制得睡莲花提取物。
所述睡莲花指睡莲花植物未开放或初开的花蕾、花瓣、花蕊等。
本发明采用的原料应为睡莲花干制品,指新鲜睡莲花或普通晒干睡莲花经热风干燥至水分含量不超过10%的一类原料。原料可进行粉碎预处理,也可不经过粉碎预处理。优选的,对睡莲花干制品进行粉碎,有利于活性物质的溶出。
可用于本发明的睡莲花品种没有特别限制,可以是不同品种的睡莲花。
本发明研究表明,睡莲花提取物具有延缓皮肤衰老、抗皮肤氧化、去皱等有益护肤功效,进一步对睡莲花中护肤功效成分进行鉴定,明确睡莲花提取物中的主要活性成分包括:鞣花酸、异槲皮苷、没食子酸、樱桃苷、紫云英苷、阿福豆苷。其中樱桃苷、阿福豆苷首次在睡莲花中被发现。为了提高提取物中有效活性成分含量,本发明以成分性质为指导对提取方法进行优化。
步骤(1)中,通过控制料液比、提取温度以及原料含水量,综合提高睡莲花提取物中总黄酮、总酚的含量,实现较高的提取率。
作为优选,所述有机溶剂为乙醇、甲醇或正丁醇,有机溶剂水溶液的体积比浓度为65%~80%。在所述有机溶剂水溶液条件下能有效提取上述活性成分。
作为优选,所述睡莲花干制品的含水量≤10%,含水量控制在10%以下,有利于提高提取物中活性成分的含量。
作为优选,料液比为1g:40mL。
浸提温度对目标成分尤其是黄酮类成分的提取影响较大,作为优选,浸提的温度为45~60℃。
作为优选,浸提的时间为20~30h。
作为优选,浸提过程中结合超声处理,超声条件为:超声频率300~500kHz,超声强度5~15W/cm,时间为1.5~3.0h,超声结束后静置。
步骤(2)中,利用常见的碱性剂(如碳酸盐类)调节萃取液的pH值至7~9,本发明研究表明,鞣花酸是发挥抗皱功效的主要成分,在提取过程中,调节萃取液在碱性条件(pH=7~9)下能够增加鞣花酸、没食子酸溶解度,提高提取物中这两类成分的含量。
作为优选,利用碱性剂将萃取液的pH值至8±0.3。所述碱性剂包括但不限于氢氧化钠、碳酸钠、碳酸氢钠、碳酸氢三钠、碳酸钾、碳酸氢钾等碳酸盐类。
调节pH后,利用减压蒸发去除有机溶剂,得到浓缩液,再经冷冻干燥后得到睡莲花提取物。
本发明提供了一种由上述方法制备得到的睡莲花提取物。所述睡莲花提取物的活性成分以鞣花酸、异槲皮苷、没食子酸、樱桃苷、紫云英苷、阿福豆苷为主。
在上述条件下制备的睡莲花提取物中总黄酮重量含量为20%~60%,总酚含量为50%~80%。其中鞣花酸的重量含量为3.0%~10.0%;异槲皮苷的重量含量为0.3%~4.2%;没食子酸的重量含量为0.2%~9.5%;樱桃苷的重量含量为0.1%~2.4%;紫云英苷的重量含量为0.05%~1.0%;阿福豆苷的重量含量为0.05%~0.5%。
进一步的,本发明对制备的睡莲花提取物进行功效研究,由体外及体内实验验证,上述睡莲花提取物具有较强的抗氧化和抗皱能力,无论是外用或食用均能够有效发挥淡化肌肤细纹、提高皮肤弹性、减少皮肤色素沉积等护肤养颜功效,因此可用于制备相关的化妆品、食品、医药产品。
因此,本发明提供了所述睡莲花提取物在制备化妆品或功能性食品中的应用。
进一步的,所述化妆品为具有美白、抗皱或防晒功效的化妆品;所述功能性食品为具有抗皮肤老化功效的食品或饮品。
本发明还提供了一种含有本发明的睡莲花提取物作为有效成分的组合物(包括食品组合物、护肤品组合物等)。这些组合物通过外用或食用的方式用于皮肤的延缓衰老、抗氧化、去皱等。
进一步地,所述组合物由0.01~99.9wt%的睡莲花提取物和作为余量的载体组成。
所述睡莲花提取物用于制备具有抗皱、美白、防晒等效果的化妆品,化妆品的具体形式包括但不限于化妆水、喷雾、乳液、精华水、精华乳、霜、凝露、粉底液、肌底液、遮瑕膏、面霜或面膜。
所述睡莲花提取物用于制备具有抗皱等效果的健康食品或饮料,具体形式包括但不限于果蔬制品、肉蛋奶制品、焙烤食品、冷冻食品、粮食制品、水产制品、糖果及可可制品、甜味料、调味品、饮料、酒类、特膳食品等。
本发明具备的有益效果:
(1)通过本发明技术实现了睡莲花中护肤功效成分的高效富集提取,严格控制原料含水量以及浸提条件以提高提取物得率,进一步的,在浸提结束后调节萃取液pH至7~9呈碱性,本发明增加这一工艺步骤有助于提高萃取液中活性成分鞣花酸、没食子酸的溶解度,增加其含量,得到的睡莲花提取物质量好、品质稳定,并且工艺操作简便,对生产设备要求较低,利于产业化发展。
(2)本发明提供的提取物中主要成分鞣花酸和异槲皮苷含量相对高,首次报道睡莲花提取物中含有樱桃苷、阿福豆苷。该提取物具有较强的抗氧化和抗皱能力,无论是外用或食用均能够有效发挥淡化肌肤细纹、提高皮肤弹性、减少皮肤色素沉积等护肤养颜功效,在化妆品、食品、医药产品等领域中具有极大的应用前景。
附图说明
图1为睡莲花提取物总离子流图。
图2为提取物中鞣花酸母离子质谱图。
图3为提取物中鞣花酸子离子质谱图。
图4为提取物中异槲皮苷母离子质谱图。
图5为提取物中异槲皮苷子离子质谱图。
图6为提取物中没食子酸母离子质谱图。
图7为提取物中没食子酸子离子质谱图。
图8为提取物中樱桃苷母离子质谱图。
图9为提取物中樱桃苷子离子质谱图。
图10为提取物中紫云英苷母离子质谱图。
图11为提取物中紫云英苷子离子质谱图。
图12为提取物中阿福豆苷母离子质谱图。
图13为提取物中阿福豆苷子离子质谱图。
图14为上述6种化合物的结构式。
具体实施方式
下面将结合具体实施例,对本发明做进一步的详细说明。但是,这些实施例、对比例仅用于更详细地说明本发明,并不对本发明所附权利要求范围构成任何限制。
下述实施例中所使用的材料、试剂等,如无特殊说明,为可从商业途径得到的试剂和材料。原料“爱德拉多”品种睡莲花采自广西南宁,统一经40℃烘制24h以上。
以下为通用方法:
1)总黄酮含量测定:
采用硝酸铝-亚硝酸钠比色法。取稀释一定倍数的睡莲花提取物样品或芦丁标准液(0、30、60、90、120、150mg/L)2.5mL,加入150μL 5%亚硝酸钠溶液,摇匀后放置6min,再加入300μL 10%硝酸铝溶液摇匀后放置5min,加入1mL 1.0mol/L氢氧化钠溶液,用水定容至6mL,测定510nm处吸光值。分别以芦丁溶液浓度、吸光值为横、纵坐标作标准曲线,计算总黄酮含量,结果以100g睡莲花提取物中芦丁当量表示。
2)总酚含量测定:
采用福林酚法测定总酚。取稀释一定倍数的睡莲花提取物样品或绿原酸标准液(0、20、40、60、80、100mg/L)500μL,加100μL福林酚试剂,混匀后反应6min,加入1mL 7%Na2CO3,并定容至6mL,37℃下恒温反应90min后,测定750nm处吸光值。分别以绿原酸溶液浓度、吸光值为横、纵坐标作标准曲线,计算总酚含量,结果以100g睡莲花提取物中绿原酸当量表示。
3)提取物的定性、定量分析—UPLC高分辨质谱
通过UPLC-QE高分辨质谱对提取部位进行成分定性分析。测定条件为ACQUITY BEHC18柱(2.1mm×100mm,Waters)用于UPLC分析,柱温为40℃,流速为0.4mL/min,流动相为0.1%甲酸水溶液(A)和0.1%甲酸-乙腈溶液(B)。流动相的梯度程序如下:0min(A:B=95:5)、3min(A:B=75:25)、4min(A:B=35:65)和10min(A:B=35:65)。进样量为10μL。QE在正负离子模式下工作。操作参数设定为:锥电压30v,毛细管电压2kv,源温度100℃。在95~1400的质荷比(m/z)范围内记录数据,扫描时间为0.25s,扫描间隔为0.02s,持续10min。在此基础上,结合质谱数据库Compound DiscovererTM检索匹配和文献查询,确定可能的成分。对新的及含量较高的成分利用UPLC-PDA进行定量分析。
实施例1
1、制备方法:将1kg干燥的水分含量≤10%(按照GB 5009.3-2016直接干燥法进行测定,若不符合水分含量要求则继续40℃烘制干燥)的睡莲花花瓣按1g:40mL料液比,加入70%(v/v)的乙醇水溶液,控制温度为45℃,于超声频率400kHz,超声强度10W/cm2,进行浸提2h以充分萃取并静置24h。通过离心将萃取液与睡莲花沉淀物分离,使用1mol/L Na2CO3溶液调节萃取液pH为8呈碱性。通过减压蒸发回收乙醇,得到浓缩液,经冷冻干燥后即得到睡莲花提取物。
2、对所得提取物的质量以及成分进行分析,鉴定结果如图1-13所示,具体数据如表1所示。上述结果明确了鞣花酸、异槲皮苷、没食子酸、樱桃苷、紫云英苷、阿福豆苷为主要活性成分,结构式如图14所示。
实施例2
制备方法:将1kg干燥的水分含量≤10%(按照GB 5009.3-2016直接干燥法进行测定,若不符合水分含量要求则继续40℃烘制干燥)的睡莲花花瓣按1g:40mL料液比,加入65%(v/v)的甲醇水溶液,控制温度为45℃,于超声频率400kHz,超声强度10W/cm2,进行浸提2h以充分萃取并静置24h。通过过滤将萃取液与睡莲花沉淀物分离,使用1mol/L NaHCO3溶液调节pH为7。通过减压蒸发回收甲醇,得到浓缩液,经冷冻干燥后即得到睡莲花提取物。对所得提取物的质量以及成分进行分析,具体数据如表1所示。
实施例3
制备方法:将1kg干燥的水分含量≤10%(按照GB 5009.3-2016直接干燥法进行测定,若不符合水分含量要求则继续40℃烘制干燥)的睡莲花花蕾按1g:60mL料液比,加入80%(v/v)的正丁醇水溶液,控制温度为60℃,于超声频率400kHz,超声强度10W/cm2,进行浸提2h以充分萃取并静置24h。通过过滤将萃取液与睡莲花沉淀物分离,使用2mol/L NaOH溶液调节pH为9。通过减压蒸发回收正丁醇,得到浓缩液,经冷冻干燥后即得到睡莲花提取物。对所得提取物的质量以及成分进行分析,具体数据如表1所示。
对比例
1、料液比条件
对比例1-1,将实施例1中的料液比改成1g:10mL,其余同实施例1。
对比例1-2,将实施例1中的料液比改成1g:70mL,其余同实施例1。
2、温度条件
对比例2-1,将实施例1中的温度改成15℃,其余同实施例1。
对比例2-2,将实施例1中的温度改成70℃,其余同实施例1。
3、pH条件
对比例3-1,将实施例1中的pH改成6.5,其余同实施例1。
对比例3-2,将实施例1中的pH改成9.5,其余同实施例1。
4、原料含水量条件
对比例4-1,将实施例1中的水分含量改成15%,其余同实施例1。
表1不同制备方法参数下睡莲花提取物得率及各成分含量
得率单位:%;总黄酮含量单位:g芦丁当量/100g花提取物;总酚含量单位:g绿原酸当量/100g花提取物;各主要成分单位:%。
如表1所示,料液比、温度、萃取液pH值及原料含水量都会影响睡莲花提取物得率以及活性成分含量。其中,温度的影响较大,温度过高会破坏活性成分,尤其是黄酮类成分,此外鞣花酸会因受热而降解为没食子酸。而温度过低会导致提取得率低下。另外,萃取液pH在碱性条件下会增加鞣花酸、没食子酸溶解度,使得提取物中这两类成分含量更高,但pH碱性过强时,黄酮类成分含量会降低。此外,当原料睡莲花水分含量大于10%时,提取物得率会降低。
活性检测例1
通过DPPH,ABTS,ORAC,FRAP法测定实施例1,2,3中睡莲花提取物体外抗氧化能力研究,数据如表2所示。具体方法如下:
1)DPPH法:
实验前精确称取20mg的DPPH溶于适量甲醇中,定容至500mL。以25μM~800μMTrolox为对照品,取稀释一定倍数的提取物溶液或对照品溶液100μL,加入3.9mL DPPH,37℃避光反应60min,于515nm波长处测定吸光值。以Trolox浓度为横坐标,吸光值为纵坐标作标准曲线,结果以每克提取物中Trolox当量表示(mmol Trolox/g DW)。
2)ABTS法:
分别配制7.4mM ABTS以及2.6mM过硫酸钾,临用前等体积混合,37℃避光反应12h,稀释约27倍后,直至734nm下吸光值为约0.7后即可使用。以25μM~800μM Trolox为对照品,取稀释一定倍数的提取物溶液或对照品溶液100μL,加入3.0mL ABTS反应液,37℃避光反应60min后于734nm处测定吸光值。以Trolox浓度为横坐标,吸光值为纵坐标作标准曲线,结果以每克提取物中Trolox当量表示(mmol Trolox/g DW)。
3)FRAP法:
以40mM HCl为溶剂配置10mM TPTZ溶液,20mM FeCl3水溶液,30mM,pH=3.6乙酸钠缓冲液,将三者以1:1:10(v/v/v)混合制得FRAP工作液(于2h内用完)。以FeSO4为对照品,取稀释一定倍数后的提取物溶液或对照品溶液100μL,加入3.0mL FRAP工作液,37℃避光反应60min后于595nm处测定吸光值。以FeSO4浓度为横坐标,吸光值为纵坐标作标准曲线,结果以每克提取物中FeSO4当量表示(mmol FeSO4/g DW)。
4)ORAC法:
配置pH 7.4,浓度为75mM磷酸盐缓冲液,以其为溶剂配制荧光素及AAPH溶液。以0~80μM Trolox为对照品,分别向黑色酶标96孔板中加入20μL提取物溶液(稀释一定倍数后)、120μL荧光素,37℃保温5min后加入60μL AAPH,使荧光素和AAPH终浓度分别为70nM和12mM,立即进入荧光酶标仪测定。测定条件为每2min测定一次,持续3h。激发波长及发射波长分别为485nm、530nm。以Trolox浓度为横坐标,吸光值为纵坐标作标准曲线,结果以每克提取物中Trolox当量表示(mmol Trolox/g DW)。
表2不同制备方法下睡莲花提取物的体外抗氧化能力
如表2所示,以实施例1中的提取方法制备所得的睡莲花提取物体外抗氧化性最好,即通过料液比为1g:40mL,萃取处理温度为45℃,萃取液调节pH=8。
活性检测例2
睡莲花提取物及其主要成分抑制胶原蛋白酶活性研究,数据如表3所示。具体方法如下:
将10mmol/L CaCl2、400mmol/L NaCl和50mmol/L Tris-HCl缓冲液制备成备用缓冲液。用备用缓冲溶液将0.25U/mL的胶原蛋白酶、胶原蛋白酶底物leu-gly-pro-ala(FALGPA)分别配制成40μL的酶溶液和2mmol/L的底物溶液。取10μL,50mmol/L Tricine缓冲液与10μL提取物/单体成分溶液混合(将提取物或各单体成分物质用250μL DMSO溶解并用缓冲溶液配成50mg/L的母液,最终稀释成1mg/mL的溶液)。同时提取物/单体成分溶液更换为缓冲液作为空白对照,更换为EGCG作为阳性对照。混合酶溶液和样品溶液,在37℃下孵育20min。添加50μL FALGPA,立即在340nm处连续20min测量吸光度,记录0min时的吸光度与第20min时的吸光度差值为△OD,利用下列公式进行计算:
抑制率=[(对照组△OD-样品组△OD)/对照组△OD]×100%
表3睡莲花提取物及其主要成分的胶原蛋白酶抑制率
组别 | 胶原蛋白酶抑制率% |
实施例1 | 91.24 |
实施例2 | 86.34 |
实施例3 | 88.79 |
鞣花酸 | 85.11 |
异槲皮苷 | 67.21 |
没食子酸 | 76.33 |
樱桃苷 | 82.19 |
紫云英苷 | 53.88 |
阿福豆苷 | 49.57 |
阳性对照(表没食子儿茶素没食子酸酯) | 87.03 |
如表3所示,实施例1、2、3中的睡莲花提取物的胶原蛋白酶抑制率均大于85%,阳性对照表没食子儿茶素没食子酸酯(EGCG)的抑制率为87.03%。实施例1中的睡莲花提取物抑制率大于阳性对照,实施例2、3与阳性对照相近。在睡莲花提取物的主要成分中鞣花酸和樱桃苷的胶原蛋白酶抑制率大于80%,没食子酸的胶原蛋白酶抑制率大于70%,说明睡莲花提取物中发挥体外抗皱功效的成分主要是鞣花酸、樱桃苷和没食子酸。
活性检测例3
睡莲花提取物及其主要成分抑制酪氨酸酶活性研究,数据如表5所示。具体方法如下:
将提取物或各成分物质用250μL DMSO溶解并用缓冲溶液配成50mg/L的溶液,稀释成1mg/mL的溶液,按表4中反应液A1,A2,A3,A4组成,依次准备吸取等浓度的不同样品溶液和阳性对照溶液,缓冲溶液和酶溶液,充分混匀后放入37℃气浴中孵育10min,然后加入相应底物溶液,单酚酶40min后测定各反应液在475nm处的吸光值,二酚酶5min后测定各反应液在475nm处的吸光值。按照如下公式计算抑制率:
酪氨酸单酚酶/二酚酶抑制率/%=[1-(ODA3-ODA4)/(ODA1-ODA2)]×100
表4体外酪氨酸酶活性测定中的反应体系
反应液成分体系 | A<sub>1</sub>/mL | A<sub>2</sub>/mL | A<sub>3</sub>/mL | A<sub>4</sub>/mL |
目标溶液 | 0 | 0 | 0.3 | 0.3 |
缓冲溶液 | 0.4 | 0.5 | 0.1 | 0.2 |
底物(酪氨酸/L-DOPA) | 0.5 | 0.5 | 0.5 | 0.5 |
酪氨酸酶 | 0.1 | 0 | 0.1 | 0 |
表5睡莲花提取物及其主要成分对酪氨酸单酚酶和酪氨酸二酚酶的抑制率
组别 | 酪氨酸单酚酶(%) | 酪氨酸二酚酶(%) |
实施例1 | 45.19 | 48.00 |
实施例2 | 33.44 | 41.32 |
实施例3 | 28.35 | 22.88 |
鞣花酸 | 56.88 | 70.22 |
异槲皮苷 | 7.97 | 13.23 |
没食子酸 | 40.32 | 24.91 |
樱桃苷 | 23.28 | 23.41 |
紫云英苷 | 12.11 | 16.89 |
阿福豆苷 | 16.79 | 29.75 |
熊果苷(阳性对照) | 59.70 | / |
曲酸(阳性对照) | / | 96.97 |
酪氨酸酶活性可分为单酚酶(以酪氨酸为底物)和二酚酶(以多巴为底物)活性。如表5所示,实施例1、2、3中的睡莲花提取物的酪氨酸单酚酶的抑制率均大于25%,尽管弱于阳性对照熊果苷,但睡莲花提取物仍具有单酚酶抑制活性,其中主要成分鞣花酸抑制活性接近熊果苷。睡莲花提取物的酪氨酸二酚酶的抑制率均大于20%,其中主要成分鞣花酸、没食子酸、樱桃苷、阿福豆苷二酚酸抑制率均大于20%。因此,睡莲花提取物具有一定的美白功效,其主要成分鞣花酸、没食子酸、樱桃苷、阿福豆苷。
活性检测例4
睡莲花提取物及其主要成分的紫外吸收能力研究,数据如表6所示。具体方法如下:
将花卉提取物及各成分样液在290~320nm,每隔5nm测定3次吸光值,1cm石英比色皿,乙醇作为空白对照。按照如下公式计算SPF值:
表6睡莲花提取物及其主要成分的SPF值
组别 | SPF值 |
实施例1 | 17.89 |
实施例2 | 14.15 |
实施例3 | 24.45 |
鞣花酸 | 23.16 |
异槲皮苷 | 16.29 |
没食子酸 | 36.27 |
樱桃苷 | 5.68 |
紫云英苷 | 4.21 |
阿福豆苷 | 3.89 |
阳性对照(甲氧基肉桂酸乙基己酯) | 34.05 |
如表6所示,实施例1、2、3中的睡莲花提取物的SPF值分别为17.89、14.15、24.45,低于阳性对照的SPF值。表明睡莲花提取物具有一定的紫外吸收能力。在睡莲花提取物的主要成分中,没食子酸的SPF值略高于阳性对照,说明睡莲花提取物中发挥防晒功效的成分主要是没食子酸。
活性检测例5
睡莲花提取物及其主要成分细胞抗皱能力研究,数据如表7所示。具体方法如下:
细胞活力测定:人真皮成纤维细胞CCD-986sk细胞在96孔板(1×104细胞/100μL/孔)中生长一夜。然后在UVB(30mJ/cm2)照射和各样品溶液干预下处理细胞6h。使用MTS(20μL/孔)[(3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4sulfophenyl)-2H tetrazolium)]在37℃条件下孵育1h后使用酶标仪在490nm处测量吸光值。
I型胶原蛋白、基质金属蛋白酶1(MMP-1)和基质金属蛋白酶3(MMP-3)mRNA表达测定:不同样品溶液处理后,用RNAzol-B分离CCD-986sk细胞的总RNA。用随机的六脱氧核苷酸引物和逆转录酶逆转录3μL总RNA。用对应引物PCR扩增单链cDNA。PCR条件为:I型胶原蛋白,95℃变性50s,55℃退火50s,72℃延伸70s,循环30次;MMP-1,95℃变性60s,56℃退火60s,72℃延伸60s,循环30次;MMP-3,95℃变性60s,60℃退火120s,72℃延伸180s,循环30次;β-肌动蛋白,95℃变性30s,56℃退火30s,72℃延伸30s,25个循环。β-肌动蛋白作为内参评价I型胶原蛋白、MMP-1和MMP-3的相对表达。
表7睡莲花提取物及其主要成分对人真皮成纤维细胞活力、I型胶原蛋白、MMP-1、MMP-3mRNA表达的影响
*表示为UVB照射组(模型)与空白组比较,*代表P<0.05,**代表P<0.01,***代表P<0.001
#表示为各实施例与单体成分组同空白组比较,#代表P<0.05,##代表P<0.01,###代表P<0.001
实验数据显示,睡莲花提取物实施例1、2、3能维持UVB照射下人真皮成纤维细胞的活力,可能是通过增加了细胞中I型胶原蛋白表达,减少了胶原蛋白分解相关的基质金属蛋白酶MMP-1和MMP-3的表达。且这些效果优于阳性对照维生素C。因此,睡莲花提取物能够在细胞水平发挥抗皱作用。实施例1中鞣花酸含量高,上述效果更优,且单独鞣花酸组处理同样具有上述效果,效果优于阳性对照组。这些都表明睡莲花提取物中发挥抗皱功效的成分主要是鞣花酸。
活性检测例6
睡莲花提取物及其主要成分体内抗光老化能力研究,分为口服和外用干预方式,数据如表8所示。具体方法如下:
在环境适应2周后将SD大鼠随机分为10组(n=30):正常对照组(不暴露于UVB环境,以蒸馏水喂养);光老化模型组(大鼠逐渐暴露于UVB,灌胃蒸馏水);低剂量口服实施例1组(大鼠逐渐暴露于UVB,灌胃0.32g L-1提取物溶液),低剂量外抹实施例1水溶液组(大鼠逐渐暴露于UVB,涂抹100mg mL-1提取物溶液0.5mL);高剂量口服实施例1组(大鼠逐渐暴露于UVB,灌胃2.88g L-1提取物溶液),高剂量外抹实施例1水溶液组(大鼠逐渐暴露于UVB,涂抹200mg mL-1提取物溶液0.5mL);口服鞣花酸组(大鼠逐渐暴露于UVB,但灌胃0.96g L-1鞣花酸溶液),外抹鞣花酸组(大鼠逐渐暴露于UVB,但涂抹100mg mL-1鞣花酸溶液);口服茶多酚组(大鼠逐渐暴露于UVB,但灌胃0.96g L-1茶多酚溶液),外抹茶多酚组(大鼠逐渐暴露于UVB,但涂抹100mg mL-1茶多酚溶液)。
UVB治疗前,将大鼠标记区域内毛发脱落,用7%Na2S溶液作用10min进一步去除残留的毛发。除对照组外,其余组别大鼠固定在自制的面板上,背脊柱两侧暴露在自制的装置上,箱内安装UV灯。设置大鼠与灯的距离为45cm,用辐射计监测照射强度。每3d进行1次UVB照射,每次照射时间控制在10min(第1周)、15min(第2周)、20min(第3周-第18周)。UV-B的强度为70mJ cm-2。辐射结束时,测定大鼠皮肤的光老化相关指标,包括皮肤弹性、皮肤I型胶原蛋白、弹力蛋白含量、MMP-1活性。
表8睡莲花提取物及其主要成分对SD大鼠UVB诱导的皮肤光老化影响
*表示为UVB照射组(模型)与空白组比较,*代表P<0.05,**代表P<0.01,***代表P<0.001
#表示为各实施例与单体成分组同空白组比较,#代表P<0.05,##代表P<0.01,###代表P<0.001
实验数据显示,睡莲花提取物实施例1无论是在外用还是口服干预下能减少UVB照射引起的SD大鼠皮肤光老化的发生,体现在皮肤弹性增加。可能的机制是通过增加了皮肤中I型胶原蛋白、弹力蛋白表达,减少了胶原蛋白分解相关的基质金属蛋白酶MMP-1活性。且这些效果优于阳性对照茶多酚处理。因此,睡莲花提取物能够在体内水平发挥抗皮肤光老化作用。单独鞣花酸组处理同样具有上述效果,效果略优于阳性对照组。这些都表明睡莲花提取物中发挥抗皱功效的成分可能主要是鞣花酸。
应用例1护肤乳制备
鲸蜡硬脂醇醚-21:1.5%、鲸蜡硬脂醇聚醚-20:1.2%、蜡硬脂醇:3.0%、辛酸/癸酸三甘油酯:5.0%、棕榈酸异辛酯(2-EHP):4.0%、二甲基硅油(DC200):3.0%、碳酸二辛酯(CC):4.0%、尼泊金甲酯/尼泊金乙酯:0.2%/0.1%、生育酚:0.5%、睡莲花提取物(实施例1-3):0.8%、EDTA-2Na:0.1%、甘油:5%、1,3丁二醇:3.0%、黄原胶:0.25%,水:余量。
其制备方法按照如上配方,依次进行如下步骤:
(1)鲸蜡硬脂醇醚-21、鲸蜡硬脂醇聚醚-20、蜡硬脂醇、辛酸/癸酸三甘油酯、棕榈酸异辛酯(2-EHP)、二甲基硅油(DC200)、碳酸二辛酯(CC)、尼泊金甲酯/尼泊金乙酯、生育酚、睡莲花提取物组成了油相组分A,将油相组分A加热到83℃,搅拌均匀至融化,得油相原料。
(2)去离子水、EDTA-2Na、甘油、1,3丁二醇、黄原胶组成了水相组分B,将水相组分B加热至83℃,搅拌均匀至溶解,得水相原料。
(3)将83℃的油相原料加入至83℃水相原料中趁热搅拌,从而实现乳化均质(一般搅拌1min左右能实现乳化均质);继续搅冷却,室温下静置陈化24小时,得护肤乳。
最后,还需要注意的是,以上列举的仅是本发明的若干个具体实施例和对比例。显然,本发明不限于以上实施例和对比例,还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联系到的所有变形,均应认为是本发明的保护范围。
Claims (10)
1.一种睡莲花提取物的制备方法,其特征在于,包括以下步骤:
(1)将睡莲花干制品按照1g:20mL~60mL的料液比加入到体积比为50%~90%的有机溶剂水溶液中,于20~65℃条件下浸提,分离得到萃取液;
(2)调节萃取液的pH值至7~9,然后去除有机溶剂,干燥后制得睡莲花提取物。
2.如权利要求1所述的睡莲花提取物的制备方法,其特征在于,步骤(1)中,所述有机溶剂为乙醇、甲醇或正丁醇,有机溶剂水溶液的体积比浓度为65%~80%。
3.如权利要求1所述的睡莲花提取物的制备方法,其特征在于,步骤(1)中,所述睡莲花干制品的含水量≤10%,料液比为1g:40mL。
4.如权利要求1所述的睡莲花提取物的制备方法,其特征在于,步骤(1)中,浸提的温度为45~60℃,浸提的时间为20~30h。
5.如权利要求1所述的睡莲花提取物的制备方法,其特征在于,步骤(1)中,浸提过程中结合超声处理,超声条件为:超声频率300~500kHz,超声强度5~15W/cm,时间为1.5~3.0h,超声结束后静置。
6.如权利要求1所述的睡莲花提取物的制备方法,其特征在于,步骤(2)中,利用碱性剂将萃取液的pH值至8±0.3;所述碱性剂为氢氧化钠、碳酸钠、碳酸氢钠、碳酸氢三钠、碳酸钾或碳酸氢钾。
7.如权利要求1-6任一项所述的制备方法制得的睡莲花提取物。
8.如权利要求7所述的睡莲花提取物,其特征在于,其组成包括鞣花酸、异槲皮苷、没食子酸、樱桃苷、紫云英苷和阿福豆苷。
9.如权利要求7或8所述的睡莲花提取物在制备化妆品或功能性食品中的应用。
10.如权利要求9所述的应用,所述化妆品为具有抗皱、美白或防晒功效的化妆品;所述功能性食品为具有抗皮肤老化功效的食品或饮品。
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