CN115487228A - 一种芍药花提取物及其提取方法、检测方法和应用 - Google Patents
一种芍药花提取物及其提取方法、检测方法和应用 Download PDFInfo
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- CN115487228A CN115487228A CN202211114956.5A CN202211114956A CN115487228A CN 115487228 A CN115487228 A CN 115487228A CN 202211114956 A CN202211114956 A CN 202211114956A CN 115487228 A CN115487228 A CN 115487228A
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- peony
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Abstract
本发明公开了一种芍药花提取物及其提取方法、检测方法和应用,属于芍药花提取技术领域。芍药花提取物包括以下组分:没食子酸的含量范围为57.54‑1540.7μg/ml;氧化芍药苷的含量范围0‑97μg/ml;芍药内酯苷的含量范围9.06‑251.32μg/ml;芍药苷的含量范围61.71‑447.26μg/ml;芦丁的含量范围0.61‑2497.30μg/ml;五没食子酰葡萄糖的含量范围72.02‑1566.94μg/ml;苯甲酰芍药苷的含量范围0‑491.91μg/ml,本发明得到的芍药花提取物具有较多的活性成分。
Description
技术领域
本发明涉及芍药花提取技术领域,更具体的涉及一种芍药花提取物及其提取方法、检测方法和应用。
背景技术
芍药(Paeoniae Alba Radix)为毛茛科,芍药属植物。世界约30种,分布于北温带,大部产亚洲,部分种类分布在欧洲南部和北美洲西部。中国有11种,主要分布西南、西北地区,其各种培植变种繁多。芍药在我国分布广泛,在东北480-800m海拔的山坡、草地、林下及河北、山西、内蒙古、陕西及甘肃、宁夏、四川等地海拔1000-2300m山坡草地均有分布。芍药多生长于山川河谷地带或土丘陵墓之上,主产于浙江、安徽、四川、山东、贵州、河南、云南等地。芍药作为中国传统名花之一,具悠久的栽培历史,作为观赏植物栽培,最早见于晋代崔豹的《古今注》中,被列为中国六大名花之一,且位列草本之首,其被人们誉为“花仙”和“花相”,又被称为“五月花神”。另外,芍药在红楼梦中是一种重要的花,史湘云醉眠芍药茵是红楼梦中最美丽的情景之一,也是安徽省亳州市的市花。因此,芍药花在我国具有重要地位。
芍药含有丰富的化学成分。主要包括单萜苷类、三萜及其苷类、挥发油类、多糖类、黄酮类、鞣质类、生物碱、甾醇和氨基酸等多种化学成分。研究表明,芍药具有抗菌、抑菌、抗炎、抗氧化、镇痛镇静、抗惊厥、保护神经元、降血脂等药理作用。此外,芍药提取物能从多途径抑制自身免疫反应。临床上用于类风湿关节炎、膝关节骨关节炎、强制性脊柱炎、系统性红斑狼疮、内因性葡萄膜炎以及急、慢性肝炎、肝硬化等疾病的治疗。芍药花作为芍药生长发育的器官,含有丰富的化合物,包括多种氨基酸、单萜苷类、三萜及其苷类、挥发油类、多糖类、黄酮类、鞣质类、生物碱、甾醇和氨基酸等多种化学成分。具有丰富的生物活性,包括抗氧化、活血化瘀、益气生精。且芍药花无毒故可食用,可作为药食同源植物。目前,国内外对于芍药花的研究有限。因此,基于芍药花开发功能产品可以满足人们对于健康的需求,前景广阔。且芍药花在种植芍药的过程中却被完全忽视,任由其花开花谢,造成巨大的资源浪费。因此,项目研发的相关产品,实现芍药花资源的充分利用,对芍药花的综合开发具有重要意义。
芍药作为中国传统中药材,其花也是中国名花之一,具悠久的栽培历史。芍药花中含有丰富的化学物质。研究表明,芍药花富含蛋白质、氨基酸、多糖类、锌铁铜等微量元素、有机酸类等多种营养成分,此外还含有丰富的单萜苷类、三萜及其苷类、酚类化合物具有抑菌、抗炎、抗氧化、活血化瘀、益气生精等功效。
近年来,随着经济发展,人们消费水平日渐提高,人们越来越关注自己的身体健康问题。其中皮肤作为人体最大的器官,改善皮肤的健康问题显得越来越重要。改善肌肤状况,以到达永葆青春的目的。而芍药是我国传统中药,具有益气生精、活血化瘀等众多药理活性。其花也具有重要作用。
然而,目前市场上以芍药花提取物为主要活性成分的相关产品并不多,且通过调研发现,在药用芍药的栽培过程中,为保证根部养分供给而采取打花措施,造成芍药花资源的流失。
专利CN11437695A在提取芍药花提取物时,主要采用的是芍药干花 30~60℃条件下浸提,提取时间20~30h。芍药花在干燥过程中,由于环境的变化以及长时间的脱水处理,花瓣中所含的物质会被分解,从而导致其得到的提取物包含的活性成分较少。
发明内容
针对以上问题,本发明提供了一种芍药花提取物,提取的芍药花提取物具有较多的活性成分,包括没食子酸、氧化芍药苷、芍药苷、芍药内酯苷、1,2,3,4,6- 五没食子酰葡萄糖、芦丁、苯甲酰芍药苷。
本发明的第一个目的是提供一种芍药花提取物,所述芍药花提取物包括以下组分:没食子酸的含量范围为57.54-1540.7μg/ml;氧化芍药苷的含量范围 0-97μg/ml;芍药内酯苷的含量范围9.06-251.32μg/ml;芍药苷的含量范围 61.71-447.26μg/ml;芦丁的含量范围0.61-2497.30μg/ml;五没食子酰葡萄糖的含量范围72.02-1566.94μg/ml;苯甲酰芍药苷的含量范围0-491.91μg/ml。
本发明的第二个目的是提供上述芍药花提取物的制备方法,将芍药鲜花加入到提取溶剂中,提取3-4h后,过滤得到芍药花提取物,其中,提取溶剂为超纯水、体积分数30%的乙醇、体积分数50%的乙醇、体积分数70%的乙醇、体积分数90%的乙醇、无水乙醇中的一种。
优选的,芍药鲜花与提取溶剂的比例为3g:20-50ml。
优选的,提取方法为:在85-90℃加热3-4h后,停止加热,恢复至室温用提取溶剂恒重后过滤。
优选的,提取方法为:在2200-2500W功率下超声3-4h后,停止超声,恢复至室温用提取溶剂恒重后过滤。
本发明的第三个目的是提供上述芍药花提取物在制备抗氧化产品中的应用。
本发明的第四个目的是提供上述芍药花提取物的检测方法,按照以下步骤进行检测:
分别取没食子酸、氧化芍药苷、芍药苷、芍药内酯苷、1,2,3,4,6-五没食子酰葡萄糖、芦丁、苯甲酰芍药苷的标准品溶液制备成混合标准品溶液;采用 shim-packC18色谱柱,以乙腈为流动相A,0.5%体积的醋酸水为流动相B进行梯度洗脱,柱温30℃,调整总流速为0.8mL/min;检测波长230nm;吸取混合标准品溶液和上述芍药花提取物20μL注入液相色谱仪,测定,计算,即得。
优选的,洗脱程序:0-10min90%B;10-15min90-88%B;15-20min88-86%B; 20-25min86-83%B;25-35min83-80%B;35-40min80-78min%B;40-50min 78-77%B;50-60min77-40%B。
优选的,没食子酸、氧化芍药苷、芍药苷、芍药内酯苷、1,2,3,4,6-五没食子酰葡萄糖、芦丁、苯甲酰芍药苷的标准品溶液浓度分别为1.34mg/ml、 1.48mg/ml、3.55mg/ml、1.84mg/ml、4.14mg/ml、1.61mg/ml;
混合标准品溶液由浓度为167.5μg/ml的食子酸、185μg/ml的氧化芍药苷、 443μg/ml的芍药内酯苷、230μg/ml的芍药苷、517μg/ml的芦丁、443μg/ml的1, 2,3,4,6-五没食子酸酰葡萄糖、201μg/ml的苯甲酰芍药苷制备得到。
与现有技术相比,本发明具有以下有益效果:
成熟的花瓣细胞中含有85%的水,因此,本发明以水为主要溶剂,增加适量的乙醇作为溶剂,或者是以无水乙醇作为溶剂,采用超声提取法或热回流提取法进行提取,得到了芍药花提取物,芍药花提取物包括没食子酸、氧化芍药苷、芍药苷、芍药内酯苷、1,2,3,4,6-五没食子酰葡萄糖、芦丁、苯甲酰芍药苷。
本发明采用超声提取法,热回流提取法以及不同溶剂比例提取了不同活性成分的芍药花提取物。结果表明,热回流提取法相对于超声提取法对活性成分的提取效果较好,且不同浓度的提取溶剂对不同活性成分的提取量不同。并且本发明主要针对的是对于后期提取物的应用上。因此在比较了不同提取方法后,综合考虑选择了以30%乙醇为主要提取溶剂进行提取。
本发明提取得到的芍药花提取物具有良好的抗氧化效果,可用于生产相关产品,不仅能够带动相关产业的发展,还能为药农带来一定经济收益。同时,可以增加芍药花野生资源的综合利用。
附图说明
图1为本发明的技术路线图;
图2为不同浓度乙醇通过热回流提取法对样品提取结果指纹图谱比对;
图3为不同浓度乙醇通过超声提取法对样品提取结果指纹图谱比对;
图4为不同料液比以30%乙醇进行热回流提取法对样品提取结果指纹图谱比对;
图5为不同溶剂热回流提取法对ABTS自由基清除的影响;
图6为不同溶剂超声提取法对ABTS自由基清除的影响;
图7为不同溶剂热回流提取法对DPPH自由基清除的影响;
图8为不同溶剂超声提取法对DPPH自由基清除的影响;
图9为Fe2+标准曲线图;
图10为不同溶剂热回流提取法总还原力的测定;
图11为不同溶剂超声提取法总还原力的测定;
图12为不同溶剂热回流提取物对酪氨酸酶活力影响。
具体实施方式
下面将结合本发明实施例中的附图,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。
下列实施例中未注明具体条件的试验方法,通常按照常规条件,所述试剂和材料,如无特殊说明,均可在市场上购买得到。
实施例1
精密称取当地药农种植的芍药鲜花50g进行提取,具体的,将芍药鲜花按料液比3g:20ml加入到超纯水,于85℃恒温回流加热3h后,停止加热,恢复至室温用提取溶剂恒重后过滤,所得提取液即为芍药花提取物,作为检测液进行后续检测。
实施例2
精密称取芍药鲜花50g进行提取,具体的,将芍药鲜花按料液比3g:20ml 加入体积分数为30%的乙醇,于85℃恒温回流加热3h后,停止加热,恢复至室温用提取溶剂恒重后过滤,所得提取液即为芍药花提取物,作为检测液进行后续检测。
实施例3
精密称取芍药鲜花50g进行提取,具体的,将芍药鲜花按料液比3g:20ml 加入体积分数为50%的乙醇,于85℃恒温回流加热3h后,停止加热,恢复至室温用提取溶剂恒重后过滤,所得提取液即为芍药花提取物,作为检测液进行后续检测。
实施例4
精密称取芍药鲜花50g进行提取,具体的,将芍药鲜花按料液比3g:20ml 加入体积分数为70%的乙醇,于85℃恒温回流加热3h后,停止加热,恢复至室温用提取溶剂恒重后过滤,所得提取液即为芍药花提取物,作为检测液进行后续检测。
实施例5
精密称取芍药鲜花50g进行提取,具体的,将芍药鲜花按料液比3g:20ml 加入体积分数为90%的乙醇,于85℃恒温回流加热3h后,停止加热,恢复至室温用提取溶剂恒重后过滤,所得提取液即为芍药花提取物,作为检测液进行后续检测。
实施例6
精密称取芍药鲜花50g进行提取,具体的,将芍药鲜花按料液比3g:20ml 加入无水乙醇,于85℃恒温回流加热3h后,停止加热,恢复至室温用提取溶剂恒重后过滤,所得提取液即为芍药花提取物,作为检测液进行后续检测。
实施例7
精密称取芍药鲜花50g进行提取,具体的,将芍药鲜花按料液比3g:20ml 加入超纯水,常温下,超声功率2200W超声3h后,停止超声,恢复至室温用提取溶剂恒重后过滤,所得提取液即为芍药花提取物,作为检测液进行后续检测。
实施例8
精密称取芍药鲜花50g进行提取,具体的,将芍药鲜花按料液比3g:20ml 加入体积分数为30%的乙醇,常温下,超声功率2200W超声3h后,停止超声,恢复至室温用提取溶剂恒重后过滤,所得提取液即为芍药花提取物,作为检测液进行后续检测。
实施例9
精密称取芍药鲜花50g进行提取,具体的,将芍药鲜花按料液比3g:20ml 加入体积分数为50%的乙醇,常温下,超声功率2200W超声3h后,停止超声,恢复至室温用提取溶剂恒重后过滤,所得提取液即为芍药花提取物,作为检测液进行后续检测。
实施例10
精密称取芍药鲜花50g进行提取,具体的,将芍药鲜花按料液比3g:20ml 加入体积分数为70%的乙醇,常温下,超声功率2200W超声3h后,停止超声,恢复至室温用提取溶剂恒重后过滤,所得提取液即为芍药花提取物,作为检测液进行后续检测。
实施例11
精密称取芍药鲜花50g进行提取,具体的,将芍药鲜花按料液比3g:20ml 加入体积分数为90%的乙醇,常温下,超声功率2200W超声3h后,停止超声,恢复至室温用提取溶剂恒重后过滤,所得提取液即为芍药花提取物,作为检测液进行后续检测。
实施例12
精密称取芍药鲜花50g进行提取,具体的,将芍药鲜花按料液比3g:20ml 加入无水乙醇,常温下,超声功率2200W超声3h后,停止超声,恢复至室温用提取溶剂恒重后过滤,所得提取液即为芍药花提取物,作为检测液进行后续检测。
实施例13
精密称取芍药鲜花50g进行提取,具体的,将芍药鲜花按料液比3g:40ml 加入体积分数为30%的乙醇,于85℃恒温回流加热3h后,停止加热,恢复至室温用提取溶剂恒重后过滤,所得提取液即为芍药花提取物,作为检测液进行后续检测。
实施例14
精密称取芍药鲜花50g进行提取,具体的,将芍药鲜花按料液比3g:50ml 加入体积分数为30%的乙醇,于85℃恒温回流加热3h后,停止加热,恢复至室温用提取溶剂恒重后过滤,所得提取液即为芍药花提取物,作为检测液进行后续检测。
实施例15
精密称取芍药鲜花进行提取,具体的,将芍药鲜花按料液比3g:20ml加入体积分数为30%的乙醇,常温下,超声功率2300W超声3.5h后,停止超声,恢复至室温用提取溶剂恒重后过滤,得到提取液即为芍药花提取物。
实施例16
精密称取芍药鲜花进行提取,具体的,将芍药鲜花按料液比3g:20ml加入体积分数为30%的乙醇,常温下,超声功率2500W超声4h后,停止超声,恢复至室温用提取溶剂恒重后过滤,得到提取液即为芍药花提取物。
实施例17
精密称取芍药鲜花50g进行提取,具体的,将芍药鲜花按料液比3g:20ml 加入体积分数为50%的乙醇,于90℃恒温回流加热4h后,停止加热,恢复至室温用提取溶剂恒重后过滤,所得提取液即为芍药花提取物,作为检测液进行后续检测。
实施例18
精密称取芍药鲜花进行提取,具体的,将芍药鲜花按料液比3g:20ml加入体积分数为50%的乙醇,于87℃恒温回流加热3.5h后,停止加热,恢复至室温用提取溶剂恒重后过滤,所得提取液即为芍药花提取物,作为检测液进行后续检测。
对比例1
精密称取芍药鲜花50g进行提取,具体的,将芍药鲜花按料液比3g:20ml 加入体积分数为30%的乙醇,于室温浸提3h后,停止加热,用提取溶剂恒重后过滤,所得提取液即为芍药花提取物,作为检测液进行后续检测。
下面对实施例1-12制备得到的芍药花提取物进行检测,如图1所示,对芍药花中主要活性成分进行定量分析,明确指出芍药花中含有以下成分,分别为没食子酸、氧化芍药苷、芍药苷、芍药内酯苷、1,2,3,4,6-五没食子酰葡萄糖、芦丁、苯甲酰芍药苷。结构式见下:
(1)标准品溶液的制备
没食子酸:精密称取没食子酸标准品13.4mg溶于10ml甲醇,得到 1.34mg/ml的储备液;
氧化芍药苷:精密称取14.8mg氧化芍药苷溶于10ml甲醇中,得到 1.48mg/ml氧化芍药苷标样;
芍药内酯苷:精密称取芍药内酯苷35.5mg于试管中,加入10ml甲醇得到 3.55mg/ml的储备液;
芍药苷:精密称取芍药苷18.4mg于试管中,加入10ml甲醇得到1.84mg/ml 的储备液;
1,2,3,4,6-五没食子酸酰葡萄糖:精密称取1,2,3,4,6-五没食子酸酰葡萄糖35.5mg,溶于10ml甲醇中,得到3.35五没食子酰葡萄糖标准品;
芦丁:精密称取41.4mg于10ml容量瓶中以甲醇定容,得到4.14mg/ml的储备液。
苯甲酰芍药苷:精密称取苯甲酰芍药苷16.1mg于10ml容量瓶中,以甲醇为溶剂定容至刻度,得到浓度为1.61mg/ml的储备液。
混合标准品溶液的配制:准确量取上述7种标准品贮备液各0.2ml至2ml 道夫管中,混匀,得到浓度分别为食子酸167.5μg/ml、氧化芍药苷185μg/ml、芍药内酯苷443μg/ml、芍药苷230μg/ml、芦丁517μg/ml、1,2,3,4,6-五没食子酸酰葡萄糖443μg/ml、苯甲酰芍药苷201μg/ml的混合标准品溶液。
(2)色谱条件
采用shim-packC18色谱柱,以乙腈(A)-0.5%醋酸水(B)为流动相,设置柱温30℃,调整总流速为0.8mL/min;检测波长230nm;梯度洗脱程序见表1。
表1 HPLC梯度洗脱时间程序
时间 | 乙腈 | 0.5%醋酸水 |
0min | 10 | 90 |
10min | 10 | 90 |
15min | 12 | 88 |
20min | 14 | 86 |
25min | 17 | 83 |
35min | 20 | 80 |
40min | 22 | 78 |
50min | 23 | 77 |
60min | 60 | 40 |
70min | Stop |
(3)线性关系
取上述混合标准品溶液1.0ml、0.8ml、0.6ml、0.2ml、0.1ml至10ml容量瓶中,以甲醇为溶剂配制成5个梯度溶液。按上述(2)中的色谱条件每个梯度重复进样3次,记录各标准品的出峰时间统计各物质的峰面积,以各标准品浓度(x)为横坐标,峰面积(y)为纵坐标绘制标准曲线。结果表明没食子酸在 1.68-167.50μg/ml、氧化芍药苷在1.85-185.21μg/ml、芍药内酯苷在4.50-443.75 μg/ml、芍药苷在2.32-230.25μg/ml、芦丁在2.18-517.20μg/ml、1,2,3,4,6-五没食子酰葡萄糖在4.19-418.75μg/ml、苯甲酰芍药苷在2.12-200.23μg/ml 范围内具有良好的线性关系。各标准品线性关系,结果见表2。
表2各标准品标准曲线
成分 | 回归方程 | 相关系数R2 | 线性范围μg/ml |
没食子酸 | y=37473x+97936 | 0.9966 | 1.68-167.50 |
氧化芍药苷 | y=35815x+21014 | 0.9988 | 1.85-185.21 |
芍药内酯苷 | y=26611x+218919 | 0.9974 | 4.50-443.75 |
芍药苷 | y=28603x+15845 | 0.9989 | 2.32-230.25 |
芦丁 | y=35838x+169006 | 0.9989 | 2.18-517.20 |
1,2,3,4,6-五没食子酰葡萄糖 | y=46604x-111432 | 0.9996 | 4.19-418.75 |
苯甲酰芍药苷 | y=44541x+52981 | 0.9997 | 2.12-200.23 |
(4)含量测定
对上述各样品进行检测,取上述制备的供试品溶液,按色谱条件进样测定,进样量20μL,重复进样3次,记录出峰时间以及峰面积,计算各成分含量。没食子酸、氧化芍药苷、芍药内酯苷、芍药苷、芦丁、五没食子酰葡萄糖、苯甲酰芍药苷的含量。各提取方法中,各物质的含量,结果见表3。
表3各提取方法芍药花各物质含量
表3中,30醇、50醇、70醇、90醇分别指体积分数为30%、50%、70%、 90的乙醇,无水指无水乙醇。图2为不同浓度乙醇通过热回流提取法对样品提取结果指纹图谱比对,图3为不同浓度乙醇通过超声提取法对样品提取结果指纹图谱比对,其中,图2-3中纯水指溶剂为超纯水,30%乙醇指溶剂为体积分数为30%的乙醇,50%乙醇指溶剂为体积分数为50%的乙醇,70%乙醇指溶剂为体积分数为70%的乙醇,90%乙醇指溶剂为体积分数为90%的乙醇,无水乙醇指溶剂为无水乙醇。图4为不同料液比芍药花在30%乙醇为溶剂通过热回流法对样品提取结果指纹图谱比对,其中,图4中的1:20对应的是芍药鲜花与溶剂的料液比3g:20ml,1:40对应的是芍药鲜花与溶剂的料液比3g:40ml,1:50 对应的是芍药鲜花与溶剂的料液比3g:50ml。
由表3和图2-4可知,不同提取方法以及料液比对芍药花中不同成分的含量不同。其中,没食子酸的含量范围57.54-1540.7μg/ml。氧化芍药苷的含量范围0-97μg/ml。芍药内酯苷的含量范围9.06-251.32μg/ml。芍药苷的含量范围 61.71-447.26μg/ml。芦丁的含量范围0.61-2497.30μg/ml。五没食子酰葡萄糖的含量范围72.02-1566.94μg/ml。苯甲酰芍药苷的含量范围0-491.91μg/ml。浸提法提取得到的芍药花提取物的活性成分较少,仅含有没食子酸、芍药内酯苷、芍药苷、五没食子酰葡萄糖,进一步证明了浸提法无法提取得到含较多活性成分的芍药花提取物。
因此,作为芍药质量控制,标准选择以30%乙醇为溶剂,芍药鲜花和提取溶剂按照料液比3g:20ml提取芍药花活性成分。依据上述实验结果,要求没食子酸含量不低于498.62μg/ml,氧化芍药苷含量不低于11.82μg/ml,芍药内酯苷含量不低于214.72μg/ml,芍药苷含量不低于143.04μg/ml,芦丁含量不低于 16.60μg/ml。五没食子酰葡萄糖含量不低于1566.94μg/ml,苯甲酰芍药苷不低于59.87μg/ml。
下面对制备得到的芍药花提取物进行抗氧化测试。
(1)供试样品溶液的配制
将上述制备的芍药花样品,在HPLC色谱条件下分析,结合线性关系,计算含量,以芍药苷含量为恒定标准。将各样本调整至一致。
(2)自由基工作液的配制
DPPH工作液的配制:精密称取3.94mg DPPH定容至10ml无水乙醇中。取1ml上述溶液定容至10ml,得到0.1mM DPPH溶液,使用前调整工作液吸光值至0.7±0.02。
FRAP工作液的配制:
300mM醋酸缓冲溶液的配制:精密称取1.869g醋酸钠粉末,加16ml醋酸以水为溶剂定容至1L。
10mM TPTZ溶液的配制:精密称取0.312g TPTZ粉末,加入0.34ml浓盐酸,以水为溶剂定容至100ml。
20mM三氯化铁溶液的配制:精密称取2.703g三氯化铁粉末,以水为溶剂定容至500ml。使用时将上述三种液体按照醋酸缓冲溶液:TPTZ:三氯化铁溶液=10:1:1。
ABTS工作液的配制:分别称取ABTS标准品粉末、过硫酸钾粉末0.0384 g和0.0134g分别以水定容至10mL。将上述溶液按体积1:1混合避光反应12h,使用前调整ABTS工作液吸光值至0.7±0.02。
(3)阳性对照品的制备
精密称取维生素C(Vc)样品0.0600g加超纯水定容至10ml,得到6mg/ml 的Vc储备液。将所得VC储备液以超纯水稀释至0.06mg/ml、0.006mg/ml,其中0.006mg/ml用于ABTS、DPPH抗氧化检测,0.06mg/ml用于FRAP总还原力的测定。
(4)测试结果
1.芍药提取物ABTS自由基的清除
分别取上述不同提取方法制备的芍药花提取物,以超纯水为溶剂稀释到相同水平。分别取各供试品溶液100μL于96孔板中,加入ABTS水溶液100μL,混匀,室温避光30min,于734nm处测定吸光度。供试品溶液吸光度为A1,以纯化水作为空白对照组,吸光度为A0,以水代替ABTS溶液作为本底组,吸光度为A2。根据公式(1)计算出不同质量浓度样品溶液对ABTS自由基的清除率。各提取方法为横坐标,自由基清除率为纵坐标绘制柱状图,比较实施例1-12提取的各提取物自由基清除能力,结果见图5-6。图中0%指溶剂为超纯水,30%指溶剂为体积分数为30%的乙醇,50%指溶剂为体积分数为50%的乙醇,70%指溶剂为体积分数为70%的乙醇,90%指溶剂为体积分数为90%的乙醇,100%指溶剂为无水乙醇。
ABTS自由基清除率(%)=[1-(A1-A2)/A0]×100% (1)
2.芍药提取物DPPH自由基的清除
分别取上述制备的芍药花提取物,以超纯水为溶剂稀释到相同水平。以超纯水为溶剂稀释到相同水平。分别取各供试品溶液100μL于96孔板中,加入 DPPH乙醇溶液(0.1mmol/L)100μL,混匀,室温避光30min,于517nm处测定吸光度。供试品溶液吸光度为Ai,以纯化水作为空白对照组,吸光度为A0,以无水乙醇代替DPPH乙醇溶液作为本底组,吸光度为Aj。根据公式(2)计算出不同质量浓度样品溶液对DPPH自由基的清除率。以各提取方法为横坐标,以自由基清除率值为纵坐标绘制柱状图,比较实施例1-12提取的各提取物自由基清除能力。结果见图7-8。
DPPH自由基清除率(%)=[1-(Ai-Aj)/A0]×100% (2)
3.芍药花提取物FRAP总还原力的测定
3.1 Fe2+标准曲线的制作
在96孔板中依次加入180μL的FRAP工作液和20μL浓度为0.1014mmol/L、0.12675mmol/L、0.2535mmol/L、0.507mmol/L、1.014mmol/L的FeSO4溶液,摇匀,37℃孵育30min,测定波长为593nm的吸光度A。以吸光值为纵坐标, Fe2+浓度为横坐标绘制标准曲线,结果如图9。
3.2样品总还原力的测定
在96孔板中依次加入180μL的FRAP工作液和20μL浓度为以超纯水为溶剂将提取液稀释到相同水平。以0.06mg/ml的维生素C溶液为阳性对照。摇匀,37℃孵育30min,测定波长为593nm的吸光度A。在标准曲线上求得相应FeSO4的浓度,定义为FRAP值。FRAP值越大,抗氧化活性越强。计算总还原力,各提取方法为横坐标,以还原力值为纵坐标绘制柱状图,比较实施例1-12提取的各提取物自由基清除能力。结果见图10-11。
通过图5-11比较各提取液的DPPH、ABTS以及FRAP体外抗氧化结果。结果表明,各提取液均有较好的体外清除自由基能力。其中30%与50%乙醇提取液表现出较强的体外清除自由基能力。综上所述,以30%乙醇,按料液比 3g:20ml,热回流提取3h。为最佳的提取方法,且具有较好的清除自由基能力。
4.4酪氨酸酶活性抑制
分别取上述不同提取方法制备的芍药花提取物,以PBS为溶剂进行稀释并调节浓度,并稀释5个梯度,记为供试品溶液。分别取各供试品溶50μL不同浓度的各样品溶液、PBS溶液80μL、酪氨酸酶溶液50μL依次加入96孔板中,设5个复孔,在37℃下孵育10min,再向每个孔中加入20μL底物触发反应,孵育30min,用酶标仪测定475nm处的吸光度值,重复测定3次。记录A、B、 C、D的吸光度值。底物为左旋多巴L-dopa。需要说明的是,A、B、C、D在测试时体积均为200μL,当某一组中不添加酶或者供试品溶液时,则用PBS补充至200μL。
其中,A为空白对照组(PBS 130μL+酶50μL+底物20μL)在475nm 处测定的吸光度;B为空白背景组(PBS 180μL+底物20μL)在475nm处测定的吸光度;C为实验组(PBS 80μL+酶50μL+供试品溶液50μL+底物20 μL)在475nm处测定的吸光度;D为实验背景组(PBS 130μL+供试品溶液 50μL+底物20μL)在475nm处测定的吸光度。并计算酪氨酸酶活性抑制率,以芍药花提取物用PBS的稀释浓度(即5个梯度浓度)为横坐标,以抑制率为纵坐标绘制柱状图(图12),比较各提取物自由基清除能力。图12中水指超纯水,30%指体积分数为30%的乙醇,50%指体积分数为50%的乙醇。如图12 所示,其中体积分数为30%乙醇提取液表现出较强的酪氨酸酶活性抑制率。
尽管已描述了本发明的优选实施例,但本领域内的技术人员一旦得知了基本创造性概念,则可对这些实施例作出另外的变更和修改。所以,所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然,本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内,则本发明也意图包含这些改动和变型在内。
Claims (9)
1.一种芍药花提取物,其特征在于,所述芍药花提取物包括以下组分:没食子酸的含量为57.54-1540.7μg/ml;氧化芍药苷的含量为0-97μg/ml;芍药内酯苷的含量为9.06-251.32μg/ml;芍药苷的含量为61.71-447.26μg/ml;芦丁的含量为0.61-2497.30μg/ml;五没食子酰葡萄糖的含量为72.02-1566.94μg/ml;苯甲酰芍药苷的含量为0-491.91μg/ml。
2.一种权利要求1所述的芍药花提取物的制备方法,其特征在于,将芍药鲜花加入到提取溶剂中,提取3-4h后,过滤得到芍药花提取物,其中,提取溶剂为超纯水、体积分数为30%的乙醇、体积分数50%的乙醇、体积分数70%的乙醇、体积分数90%的乙醇、无水乙醇中的一种。
3.根据权利要求2所述的一种芍药花提取物的制备方法,其特征在于,芍药鲜花与提取溶剂的比例为3g:20-50ml。
4.根据权利要求2所述的一种芍药花提取物的制备方法,其特征在于,提取方法为:在85-90℃加热3-4h后,停止加热,恢复至室温用提取溶剂恒重后过滤。
5.根据权利要求2所述的一种芍药花提取物的制备方法,其特征在于,提取方法为:在2200-2500W功率下超声3-4h后,停止超声,恢复至室温用提取溶剂恒重后过滤。
6.一种权利要求1所述的芍药花提取物在制备抗氧化产品中的应用。
7.一种权利要求1所述的芍药花提取物的检测方法,其特征在于,按照以下步骤进行检测:
分别取没食子酸、氧化芍药苷、芍药苷、芍药内酯苷、1,2,3,4,6-五没食子酰葡萄糖、芦丁、苯甲酰芍药苷的标准品溶液制备成混合标准品溶液;采用shim-pack C18色谱柱,以乙腈为流动相A,0.5%体积的醋酸水为流动相B进行梯度洗脱,柱温30℃,调整总流速为0.8mL/min;检测波长230nm;吸取混合标准品溶液和上述芍药花提取物20μL注入液相色谱仪,测定,计算,即得。
8.根据权利要求7所述的一种芍药花提取物的检测方法,其特征在于,洗脱程序:0-10min 90%B;10-15min 90-88%B;15-20min 88-86%B;20-25min 86-83%B;25-35min83-80%B;35-40min 80-78min%B;40-50min 78-77%B;50-60min 77-40%B。
9.根据权利要求7所述的一种芍药花提取物的检测方法,其特征在于,没食子酸、氧化芍药苷、芍药苷、芍药内酯苷、1,2,3,4,6-五没食子酰葡萄糖、芦丁、苯甲酰芍药苷的标准品溶液浓度分别为1.34mg/ml、1.48mg/ml、3.55mg/ml、1.84mg/ml、4.14mg/ml、1.61mg/ml;
混合标准品溶液由浓度为167.5μg/ml的食子酸、185μg/ml的氧化芍药苷、443μg/ml的芍药内酯苷、230μg/ml的芍药苷、517μg/ml的芦丁、443μg/ml的1,2,3,4,6-五没食子酸酰葡萄糖、201μg/ml的苯甲酰芍药苷制备得到。
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