CN114376952B - 一种芍药花提取物及其制备方法和应用 - Google Patents
一种芍药花提取物及其制备方法和应用 Download PDFInfo
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- CN114376952B CN114376952B CN202111669988.7A CN202111669988A CN114376952B CN 114376952 B CN114376952 B CN 114376952B CN 202111669988 A CN202111669988 A CN 202111669988A CN 114376952 B CN114376952 B CN 114376952B
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- paeonia lactiflora
- extract
- solution
- lactiflora extract
- paeonia
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Abstract
本发明公开了一种芍药花提取物及其制备方法和应用,属于植物提取技术领域。所述芍药花提取物的制备方法包括:(1)将干燥的芍药花花蕾粉碎后,按照1g:10~30mL的料液比加入体积比为60%~90%乙醇水溶液,置于30~60℃条件下浸提制得浸提液;(2)除去浸提液中的乙醇,得到浓缩液,再用水稀释至pH为5~7,然后干燥制得所述芍药花提取物。本发明明确了芍药花中的主要成分,实现了对其中4种活性成分的高效提取,并且工艺操作简便,得到的芍药花提取物质量好、品质稳定。本发明制备的芍药花提取物具有在抗氧化、抗衰老、防晒、美白、抗皱等方面的功效,为芍药花提取物在食品、化妆品方面的应用提供了有力支持。
Description
技术领域
本发明涉及植物提取技术领域,具体涉及一种芍药花提取物及其制备方法和应用。
背景技术
芍药(Paeonia lactiflora Pall),是毛茛科芍药属的多年生草本植物,为药食兼用、且具益寿延年功用,又颇具观赏价值的草本植物,在我国作为传统中药及观赏植物栽培,其根茎可入药保健;嫩芽叶和鲜花可食用。芍药提取物为中药芍药根及花的水提或醇提物,主要活性成分为芍药苷类和没食子酸类化合物,具有扩张血管、降压镇痛、清热解痉作用,有较高的药用价值。
随着时代的发展,人们越来越重视天然产物在护肤品中的应用,以各种植物提取物作为功效成分的护肤品成为市场的热点。已有研究表明芍药花提取物具有抗氧化、抑菌、美白等方面的作用。
目前对芍药的研究多集中于芍药的根,也就是我们熟知的中药材白芍和赤芍。其实,芍药花中也富含药理活性成分,舒希凯等采用多种色谱技术分离纯化芍药花中的化学成分并采用现代波谱法鉴定其结构,结果共分离纯化出没食子酸、没食子酸甲酯、芍药苷、芍药内酯苷等11种化合物,这些酚酸类、花色苷类和黄酮类的成分,具有抗氧化、抑菌、抗衰老等功效(芍药花化学成分研究中药材,中药材,2014,37(1),66-69)。谢凯莉等建立UPLC-DAD测定白芍花瓣中的酚酸类、花色苷类、单萜苷类和黄酮类共9个有效成分的含量,其中没食子酸、芍药苷、芍药内酯苷的重量含量分别为0.011~1.188%,0.013~1.167%,0.002~1.971%,均低于2%(不同栽培品种及干燥工艺白芍花中9种有效成分含量测定及主成分分析,中国中药杂志,2020年45卷19期,4643-4651页)。
研究表明,芍药抗氧化功能的实现主要依赖于其中的五没食子酰葡萄糖,专利文献CN 112263521 A公开一种能够综合提高芍药提取物中五没食子酰葡萄糖含量的方法,具体公开:取芍药根粉碎至不大于850μm,加入体积浓度为45~55%的甲醇水溶液,加热至30~75℃回流提取30~150min得到第一提取物,再进行乙酸乙酯萃取,继而以甲醇为洗脱液进行凝胶洗脱,通过上述工艺实现五没食子酰葡萄糖的有效分离和富集。
研究表明没食子酸和1,2,3,4,6-O-五没食子酰葡萄糖在溶液中可以相互转换,但传统的芍药花提取方法中,并没有关注如何保持两者的最佳比例。此外,迄今未见有报道指出芍药花提取物在防晒和抗皱方面的功效。
发明内容
本发明的目的在于提供一种针对芍药花的活性成分进行高效提取的方法,以解决现有技术中芍药花提取物中没食子酸、芍药苷和芍药内酯苷含量低的问题。同时,对芍药花提取物的护肤功效进行深入研究,以开发其新的功能。
为实现上述目的,本发明采用如下技术方案:
本发明提供了一种芍药花提取物的制备方法,包括以下步骤:
(1)将干燥的芍药花花蕾粉碎后,按照1g:10~30mL的料液比加入体积比为60%~90%乙醇水溶液,置于30~60℃条件下浸提制得浸提液;
(2)除去浸提液中的乙醇,得到浓缩液,再用水稀释至pH为5~7,然后干燥制得所述芍药花提取物。
所述芍药花是指毛茛科植物芍药的花瓣。目前芍药花朵主要以观赏为主,本发明以芍药花瓣为原料进行活性成分提取,实现资源再利用。
本发明制备的芍药花提取物是一种以1,2,3,4,6-O-五没食子酰葡萄糖、没食子酸、芍药苷和芍药内酯苷为主的混合物。适用于本发明的芍药花品种没有特别限制,可以是不同品种的芍药。
步骤(1)中,通过控制乙醇水溶液浓度、料液比以及提取温度,综合提高芍药花提取物中总黄酮、总酚酸的含量,实现较高的提取率。
乙醇浓度低导致原料无法完全溶解使得提取率下降;乙醇浓度过高可能导致杂质析出,影响提取物的纯度。作为优选,乙醇水溶液的体积比为80%。
料液比过低,底物材料少,提取效率低;料液比过大会导致溶剂的溶解能力饱和,也会造成提取率降低。作为优选,料液比为1g:20mL。
适当升温有助于物质溶出,但温度过高会导致不稳定的成分分解。作为优选,浸提温度为40~50℃。
作为优选,浸提时间为20~30h。更为优选,浸提时间为24h。
步骤(2)中,提取物中的没食子酸和1,2,3,4,6-O-五没食子酰葡萄糖在溶液中可以相互转换,pH<6时,随着pH的升高,1,2,3,4,6-O-五没食子酰葡萄糖基本呈上升趋势,但在pH过高会导致其分解。本发明通过调节溶液pH,使得提取物中的没食子酸尽可能地转化为1,2,3,4,6-O-五没食子酰葡萄糖。作为优选,将浓缩液用水稀释至pH为6±0.3。
作为优选,利用减压蒸发除去浸提液中的乙醇。
作为优选,稀释液经减压浓缩后冷冻干燥后得到所述芍药花提取物。
本发明提供了一种由上述方法制备得到的芍药花提取物。所述芍药花提取物中的活性成分以1,2,3,4,6-O-五没食子酰葡萄糖、没食子酸、芍药苷和芍药内酯苷为主。
在上述条件下制备的芍药花提取物中总黄酮重量含量为15%~35%,总酚酸含量为60%~80%。测得提取物中的1,2,3,4,6-O-五没食子酰葡萄糖、没食子酸、芍药苷和芍药内酯苷的重量百分比含量分别为0.1~20%、1~40%、0.01~10%、0.1~10%。
作为优选,芍药花提取物中的1,2,3,4,6-O-五没食子酰葡萄糖、没食子酸、芍药苷和芍药内酯苷的含量分别为0.5~5%、10~25%、0.1~2%、0.5~5%。
进一步的,本发明对制备的芍药花提取物进行功效研究,研究表明,芍药花提取物具有抗氧化、抗衰老、淡化肌肤细纹、提高皮肤弹性、减少皮肤色素沉积以及有效抵抗紫外线等有益功效,因此可用于制备相关的食品、药物、保健品和化妆品。
本发明提供了一种含有本发明的芍药花提取物作为有效成分的组合物(包括食品组合物、药品组合物、保健品组合物、化妆品组合物和宠物饲料等)。这些组合物可用于制成具有抗氧化、抗衰老、淡化肌肤细纹、提高皮肤弹性、减少皮肤色素沉积以及有效抵抗紫外线功效的相关产品。
在获得芍药花提取物后,可用常规方法将其与食品学上、药学上、保健品或化妆品上可接受的载体、赋形剂或稀释剂相混合,形成本发明的食品组合物、药物组合物、保健品组合物和化妆品组合物。这类载体包括(但并不限于):盐水、缓冲液、葡萄糖、水、甘油、乙醇及其组合。
以药物组合物或保健品组合物为例,它们可以为固态(如颗粒剂、片剂、冻干粉、栓剂、胶囊、舌下含片)或液态(如口服液)或其他合适的形状。
本发明的活性成分(芍药花提取物)的含量通常为组合物重量的1~99%,优选为2~95%,更优选为5~90%,最佳地为10~80%。
具体的,本发明提供了所述的芍药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氧化或抗衰老功效的食品。
所述芍药花提取物包含1,2,3,4,6-O-五没食子酰葡萄糖和/或没食子酸。本发明对芍药花提取物中的单一活性成分进行功效研究,结果表明,芍药花提取物中发挥美白功效的成分主要是1,2,3,4,6-O-五没食子酰葡萄糖;发挥防晒和抗皱功效的成分主要是1,2,3,4,6-O-五没食子酰葡萄糖和没食子酸。
因此,本发明提供了1,2,3,4,6-O-五没食子酰葡萄糖和/或没食子酸在制备具有美白、抗皱或防晒功效的化妆品中的应用。
本发明具备的有益效果:
(1)本发明明确了芍药花中的主要成分,实现了对其中4种活性成分的高效提取,并且工艺操作简便,得到的芍药花提取物质量好、品质稳定。
(2)本发明制备的芍药花提取物具有在抗氧化、抗衰老、防晒、美白、抗皱等方面的功效,尤其首次公开其在防晒、抗皱方面的功效。并且明确了其中发挥防晒、美白、抗皱等护肤养颜功效的主要活性成分,为芍药花提取物在食品、化妆品方面的应用提供了有力支持。
附图说明
图1为芍药花提取物中没食子酸的质谱图(母离子)。
图2为芍药花提取物中没食子酸的质谱图(子离子)。
图3为芍药花提取物中1,2,3,4,6-O-五没食子酰葡萄糖的质谱图(母离子)。
图4为芍药花提取物中1,2,3,4,6-O-五没食子酰葡萄糖的质谱图(子离子)。
图5为芍药花提取物中芍药苷的质谱图(母离子)。
图6为芍药花提取物中芍药苷的质谱图(子离子)。
图7为芍药花提取物中芍药内酯苷的质谱图(母离子)。
图8为芍药花提取物中芍药内酯苷的质谱图(子离子)。
具体实施方式
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。
下列实施例中未注明具体条件的实验方法,通常按照常规条件,或按照制造厂商所建议的条件。
通用方法:
1、总黄酮含量测定——分光光度法:
采用硝酸铝-亚硝酸钠比色法。取稀释一定倍数的芍药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,振摇后放置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,测定吸光值A510nm。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,结果以干花中芦丁当量表示(mg RE/g DW)。
2、总酚酸含量测定——福林酚法:
以绿原酸为对照,采用福林酚法测定总酚,取500μL样品(稀释一定倍数,即每mL样品中含有2mg的芍药花提取物)或标准液(浓度为10-150μg/mL的绿原酸甲醇溶液),添加50μL 2M的福林酚,混匀反应5-8min,加入1mL 1M的NaOH溶液,用水定容至6mL,常温反应90min后760nm波长测定吸光度,计算总酚的绿原酸当量浓度。
3、提取物的定性、定量分析——UPLC高分辨质谱
首先通过UPLC高分辨质谱分析对提取部位进行成分定性分析。测定条件为ACQUITY BEH C18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的质量-电荷(m/z)范围内记录数据,扫描时间为0.25秒,扫描间隔为0.02秒,持续10分钟。在此基础上,对新的物质和含量较高的成分利用UPLC-PDA对芍药花提取物的可能功能成分进行定量分析。
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。
原料芍药来自河北保定芍药花基地。
实施例1
1、提取方法
将1kg干燥的芍药花花蕾粉碎,按照1g:20mL的料液比,加入80%(v/v)的乙醇水溶液,控制温度为50℃,静置24h;将提取液与芍药花粉末分离,通过减压蒸发回收乙醇,得到浓缩液,再将浓缩液用水稀释,直至所得稀释液的pH为6,将稀释液经冷冻干燥后即得到芍药花提取物362.7g。
2、对提取物进行成分分析,结果如表1和图1所示,明确了1,2,3,4,6-O-五没食子酰葡萄糖、没食子酸、芍药苷和芍药内酯苷为主要活性成分,其结构式分别如式(Ⅰ)-(Ⅳ)所示:
对比例1
1、料液比条件
对比例1-1,将实施例1中料液比改成1g:5mL,其余同实施例1。
最终可得到芍药花提取物285.1g。
对比例1-2,将实施例1中的料液比改成1g:50mL,其余同实施例1。
最终可得到芍药花提取物234.3g。
2、温度条件
对比例2-1,将实施例1中的温度改成20℃,其余同实施例1。最终可得到芍药花提取物201.5g。
对比例2-2,将实施例1中的温度改成70℃,其余同实施例1。最终可得到芍药花提取物214.7g。
3、pH条件
对比例3-1,将实施例1中的pH改成3,其余同实施例1。最终可得到芍药花提取物233.2g。
对比例3-2,将实施例1中的pH改成9,其余同实施例1。最终可得到芍药花提取物241.6g。
对上述各对比例的提取物进行成分分析,结果如表1所示。
表1
如表1所示,料液比、温度以及pH值都会影响芍药花提取率以及活性成分含量,其中,温度的影响最大,温度过高会破坏活性成分,温度过低导致提取效率低下。另外,pH在3左右时会导致1,2,3,4,6-O-五没食子酰葡萄糖转变成没食子酸;而碱性条件会使这些活性物质分解,不利于提取。
实施例2
1、提取方法
将1kg干燥的芍药花花蕾按照1g:20mL的料液比,加入80%(v/v)的乙醇水溶液,控制温度为50℃,静置24h;将提取液与芍药花粉末分离,通过减压蒸发回收乙醇,得到浓缩液,再将浓缩液用水稀释,直至所得稀释液的pH为7,将稀释液经冷冻干燥后即得到芍药花提取物358.2g。
2、对提取物进行成分分析,结果如表2所示。
实施例3
1、提取方法
将1kg干燥的芍药花花蕾按照1g:20mL的料液比,加入80%(v/v)的乙醇水溶液,控制温度为50℃,静置24h;将提取液与芍药花粉末分离,通过减压蒸发回收乙醇,得到浓缩液,再将浓缩液用水稀释,直至所得稀释液的pH为5,将稀释液经冷冻干燥后即得到芍药花提取物349.6g。
2、对提取物进行成分分析,结果如表2所示。
表2
如表2所示,实施例1、2、3由于pH值的不同导致了提取物中的活性成分含量不同,其中实施例2、3的1,2,3,4,6-O-五没食子酰葡萄糖含量降低,没食子酸含量升高,说明在pH=5或7时,都有一部分1,2,3,4,6-O-五没食子酰葡萄糖转换成为没食子酸。
检测例1芍药花提取物清除自由基能力
(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25-800μMTrolox为对照品,取稀释一定倍数的样品或对照品溶液100μL,加入3.9mL DPPH,37℃避光反应60min,于515nm波长处测吸光度。以Trolox浓度为横坐标,吸光值为纵坐标作标准曲线,根据标准曲线计算样品抗氧化能力,结果以每克提取物中Trolox当量表示(mmol Trolox/gDW)。
(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27倍后使用。以25-800μM Trolox为对照品,取稀释一定倍数的样品或对照品溶液100μL,加入3.0mL ABTS反应液,37℃避光反应60min后于734nm处测吸光度。以Trolox浓度为横坐标,吸光值为纵坐标作标准曲线,根据标准曲线计算样品抗氧化能力,结果以每克提取物中Trolox当量表示(mmol Trolox/g DW)。
(3)FRAP法
以40mM HCI为溶剂配置10mM TPTZ溶液,20mM FeCl3水溶液,30mM,pH=3.6乙酸钠缓冲液,三者以1:1:10(v/v/v)混合得FRAP。以FeSO4为对照品,取稀释一定倍数后的样品或对照品溶液100μL,加入3.0mL FRAP反应液,37℃避光反应60min后于595nm处测吸光度。以FeS04浓度为横坐标,吸光值为纵坐标作标准曲线,根据标准曲线计算样品抗氧化能力,结果以每克提取物中FeSO4当量表示(mmol FeSO4/g DW)。
(4)ORAC法
配置pH7.4,浓度为75mM磷酸盐缓冲液,以其为溶剂配制荧光素及AAPH溶液。以所0-80μM Trolox为对照品,分别向黑色酶标96孔板中加入20μL样品(稀释一定倍数后)、120μL荧光素,37℃保温5min后加入60μLAAPH,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、530m。以Trolox浓度为横坐标,吸光值为纵坐标作标准曲线,根据标准曲线计算样品抗氧化能力,结果以每克提取物中Trolox当量表示(mmol Trolox/g DW)。
表3
如表3所示,以实施例1中的提取方法制备所得的芍药花提取物抗氧化性最好,即通过提取时调节pH=6以保证提取物中1,2,3,4,6-O-五没食子酰葡萄糖相对含量最高,此方法提取物的抗氧化性能最好。
检测例2芍药花提取物及其主要成分抑制酪氨酸酶活性的作用研究
将各冻干溶液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,结果如表5所示。
酪氨酸酶单酚酶/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 | <![CDATA[A<sub>1</sub>/mL]]> | <![CDATA[A<sub>2</sub>/mL]]> | <![CDATA[A<sub>3</sub>/mL]]> | <![CDATA[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芍药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
酪氨酸单酚酶(%) | 酪氨酸二酚酶(%) | |
实施例1 | 86.59 | 62.49 |
实施例2 | 83.42 | 57.22 |
实施例3 | 80.35 | 52.10 |
1,2,3,4,6-O-五没食子酰葡萄糖 | 79.23 | 35.01 |
没食子酸 | 40.32 | 24.91 |
芍药苷 | 46.41 | 24.66 |
芍药内酯苷 | 42.34 | 21.72 |
曲酸(阳性对照) | 80.11 | \ |
如表5所示,实施例1、2、3中的芍药花提取物的酪氨酸单酚酶的抑制率均大于80%,高于阳性对照曲酸80.11%的抑制率,芍药花提取物酪氨酸二酚酶的抑制率高于50%。且对比其他常见花卉,芍药花的酪氨酸酶抑制率也位居前列,具有较强的美白功效。在芍药花提取物的主要成分中,1,2,3,4,6-O-五没食子酰葡萄糖的酪氨酸单酚酶抑制率和阳性对照接近,说明芍药花提取物中发挥美白功效的成分主要是1,2,3,4,6-O-五没食子酰葡萄糖。
检测例3芍药花提取物及其主要成分的紫外吸收能力研究
表6芍药花提取物及其主要成分的SPF值
SPF值 | |
实施例1 | 37.79 |
实施例2 | 35.14 |
实施例3 | 34.33 |
1,2,3,4,6-O-五没食子酰葡萄糖 | 33.96 |
没食子酸 | 36.27 |
芍药苷 | 4.67 |
芍药内酯苷 | 4.22 |
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中的芍药花提取物的SPF值为37.79,阳性对照的SPF值为34.05,高于阳性对照甲氧基肉桂酸乙基己酯,且对比其他常见花卉,芍药花的SPF最高,具有较强的紫外吸收能力。在芍药花提取物的主要成分中,1,2,3,4,6-O-五没食子酰葡萄糖和没食子酸的SPF值和阳性对照接近,说明芍药花提取物中发挥防晒功效的成分主要是1,2,3,4,6-O-五没食子酰葡萄糖和没食子酸。
检测例4芍药花提取物及其主要成分抑制胶原蛋白酶的作用研究
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、FALGPA分别配制成制40μL的酶溶液和2mmol/L的底物溶液。10μl,50mmol/LTricine缓冲液与10μL提取物混合。提取物更换为缓冲液作为空白对照,提取物更换为EGCG作为阳性对照。混合酶溶液和提取物溶液,在37℃下培养20分钟。添加50μl FALGPA(底物),立即在340nm处连续20分钟测量吸光度,记录0分钟时的吸光度与第20分钟时的吸光度差值为△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7芍药花提取物及其主要成分的胶原蛋白酶抑制率
胶原蛋白酶抑制率% | |
实施例1 | 89.62 |
实施例2 | 85.57 |
实施例3 | 82.13 |
1,2,3,4,6-O-五没食子酰葡萄糖 | 79.72 |
没食子酸 | 76.33 |
芍药苷 | 50.18 |
芍药内酯苷 | 43.96 |
阳性对照(表没食子儿茶素没食子酸酯) | 87.03 |
如表7所示,实施例1、2、3中的芍药花提取物的胶原蛋白酶抑制率均大于80%,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中的芍药花提取物抑制率大于阳性对照,实施例2、3接近阳性对照。在芍药花提取物的主要成分中,1,2,3,4,6-O-五没食子酰葡萄糖和没食子酸的胶原蛋白酶抑制率大于70%,说明芍药花提取物中发挥抗皱功效的成分主要是1,2,3,4,6-O-五没食子酰葡萄糖和没食子酸。
检测例5芍药花提取物延缓机体衰老作用研究
采用配制不同浓度溶液灌胃小鼠的方法,以观察芍药花提取物延缓机体衰老的作用。
选择88只健康昆明种小鼠,随机分为正常对照组、空白对照组、实施例1、实施例2、实施例3的芍药花提取物(500mg/kg)。造模方法采用小鼠衰老模型,以生理盐水配置10%的D-半乳糖溶液,除正常对照组外,其余6组每天在颈背部皮下分别注射0.14g/kg的D-半乳糖溶液造小鼠衰老模型,连续给予D-半乳糖50天。从第二周开始,对各实施例芍药花提取物低、高剂量组的小鼠,每天分别灌胃250mg/kg、500mg/kg的芍药花提取物;正常对照组、空白对照组小鼠灌胃等量生理盐水。每日定时观察记录小鼠的体征行为及其活动情况,记录小鼠的体重变化、毛发光泽程度、采食状况、对刺激反应的灵敏度、激怒反应、嗜睡程度等。末次给药2h后,取出脑、脾、肾、肝,吸去血液,剪去脂肪、系膜。将小鼠脾脏,经生理盐水洗净后,用滤纸粘去脏器表面水分,称重并计算脾脏指数。将脑、脾、肾、肝制备成10%的组织匀浆,按试剂盒的说明方法,分别测定小鼠的脑、脾、肾、肝中SOD和MAD含量。实验数据通过SPSS 15.0统计软件进行方差分析,结果以表示,组间采用t检验进行统计,P<0.05为差异显著。
实验结果显示,正常对照组小鼠精神状态良好、行动敏捷、毛发光泽、不易抓取;空白对照组小鼠精神不振、行动迟缓、毛色枯槁不光泽、脱毛抓取时不反抗,易于抓取。给药组小鼠上述表现较空白对照组有良性逆转;芍药花提取物各高剂量组较正常组小鼠外形无变化,活动如常。D-半乳糖空白对照组小鼠脾脏系数明显下降,与正常对照组相比有显著性差异(P<0.05),说明脾脏指数随着芍药花提取物剂量增大而明显升高。结果见表8。
正常小鼠组织中SOD含量较高,而衰老小鼠组织中SOD含量较低。芍药花提取物能够提高小鼠脑组织SOD含量,与空白组相比,低剂量组的差异无统计学意义(P>0.05),高剂量组的差异具有显著的统计学意义(P<0.01),接近正常水平。灌胃芍药花提取物后,能够提高小鼠肾组织SOD含量,与空白组相比,低剂量组差异无统计学意义(P>0.05),高剂量组的差异具有显著的统计学意义(P<0.01)。芍药花提取物能够明显提高小鼠肝组织SOD含量,与空白组相比,低、高剂量组的差异均具有显著的统计学意义(P<0.01)。具体结果见表9。
正常小鼠组织的MDA含量较低,而衰老小鼠组织的MDA含量较高。芍药花提取物能够降低小鼠脑组织MDA含量,与空白组相比,低剂量组的差异无统计学意义(P>0.05),高剂量组的差异具有显著的统计学意义(P<0.01),接近正常水平。灌胃芍药花提取物后,能够降低小鼠肾组织MDA含量,与对照组相比,低剂量组差异无统计学意义(P>0.05),高剂量组的差异具有显著的统计学意义(P<0.01)接近正常水平。芍药花提取物能够明显降低小鼠肝组织MDA含量,与空白组相比,低剂量组的差异均具有显著的统计学意义(P<0.05),高剂量组的差异具有显著的统计学意义(P<0.01),并且MDA含量低于正常水平。具体结果见表10。
表8芍药花提取物对小鼠脾脏指数的影响
注:与空白组相比,*P<0.05
表9芍药花提取物对小鼠脑、肾、肝组织SOD含量的影响
注:与空白组相比,*P<0.05,**P<0.01
表10芍药花提取物对小鼠脑、肾、肝组织MDA含量的影响
注:与空白组相比,*P<0.05,**P<0.01
实验数据显示,芍药提取物提高衰老小鼠肝组织SOD含量、降低MDA含量作用明显,说明芍药花提取物能够保护肝细胞。另外,还表明芍药花提取物能提高衰老小鼠的脾指数,能明显抑制小鼠在衰老过程中脾脏出现的萎缩,有保护免疫器官、增强免疫***的作用。这表明,芍药花提取物可提高机体免疫能力,清除体内氧自由基,抑制脂质过氧化,进而起到延缓机体衰老作用。
应用例1一种兼具美白、抗皱以及防晒功能的化妆品
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%、芍药花提取物(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、芍药花提取物组成了油相组分A,将油相组分A加热到80~85℃(较佳为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80~85℃(较佳为83℃),搅拌均匀至溶解,得水相原料。
(3)将80~85℃的油相原料加入至80~85℃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2~26小时(例如为24小时),得兼具美白、抗皱以及防晒功能的化妆品。
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 (7)
1.一种芍药花提取物的制备方法,其特征在于,包括以下步骤:
(1)将干燥的芍药花花蕾粉碎后,按照1g:10~30mL的料液比加入体积比为80%乙醇水溶液,置于50℃条件下浸提制得浸提液,浸提时间为20~30h;
(2)除去浸提液中的乙醇,得到浓缩液,再用水稀释至pH为5~7,然后经冷冻干燥后制得所述芍药花提取物;
所述芍药花提取物中的活性成分包括1,2,3,4,6-O-五没食子酰葡萄糖、没食子酸、芍药苷和芍药内酯苷,含量分别为0.5~5%、10~25%、0.1~2%、0.5~5%。
2.如权利要求1所述的芍药花提取物的制备方法,其特征在于,步骤(1)中,料液比为1g:20mL。
3.如权利要求1所述的芍药花提取物的制备方法,其特征在于,步骤(2)中,将浓缩液用水稀释至pH为6±0.3。
4.如权利要求1-3任一项所述的制备方法制得的芍药花提取物。
5.如权利要求4所述的芍药花提取物在制备化妆品或功能性食品中的应用,其特征在于,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氧化或抗衰老功效的食品。
6.一种组合物,其特征在于,包含有效剂量的如权利要求4所述的芍药花提取物。
7.如权利要求6所述的组合物,其特征在于,所述组合物中芍药花提取物的重量占比为10-80%。
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