CN113791147B - 穿破石的质量检测方法 - Google Patents
穿破石的质量检测方法 Download PDFInfo
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- CN113791147B CN113791147B CN202111008764.1A CN202111008764A CN113791147B CN 113791147 B CN113791147 B CN 113791147B CN 202111008764 A CN202111008764 A CN 202111008764A CN 113791147 B CN113791147 B CN 113791147B
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- cudrania
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Abstract
本发明公开了穿破石的质量检测方法,该方法包括以下:步骤1、穿破石供试品溶液的制备;步骤2、混合对照品溶液的制备:步骤3、标准曲线的建立:步骤4、色谱图的测定:步骤5、指纹图谱的建立。本发明根据穿破石中所含的活性成分的结构性质特点,通过大量实验筛选出最佳的流动相组成,梯度洗脱程序、流速,检测波长、色谱柱,柱温等分析条件,经多次实验验证表明,本发明提供的穿破石的含量测定方法与指纹图谱检测方法,可以全面、客观、准确的检测和评价来源为桑科柘属植物柘树的穿破石质量,为保证穿破石疗效具有重要意义。且建立的标准曲线方程,具有方法简便、稳定、精密度高、重现性好等优点。
Description
技术领域
本发明涉及一种中药的检测方法,具体涉及穿破石的质量检测方法。
背景技术
穿破石属于民间常用药、民族药和草药,出自《岭南采药录》,其来源为桑科柘属植物构棘Cudrania cochinchinensis(Lour.)Kudo et Masam或柘树Cudrania tricuspidata(Carr.)Bur et Lavallee[Maclura tricuspidata carr]的干燥根。穿破石具有祛风通络,利湿,活血通经,解毒消肿,清除湿热等功效,用于治疗风湿关节疼痛,黄疸,闭经,肺结核,胃十二指肠溃疡等,常用于急慢性肝炎的治疗,也用于治疗输卵管阻塞性不孕,糖尿病,抑制破骨细胞分化等研究。其药材标准始收录于《中国药典》1977年版一部,在多个地方标准如《湖南省中药材标准》(2009年版)、《陕西省药材标准》(2015年版)、《贵州省中药、民族药药材标准》(2019年版第一册)等中均有收录,并在多省的《中药饮片炮制规范》中也有收录。虽然穿破石在多个地方标准及省级炮制规范中有收载,但是关于穿破石的含量测定项却没有提及,在文献报道中,关于穿破石的薄层色谱法及含量测定项的相关研究较少。本发明采用高效液相色谱法和薄层色谱法建立穿破石的标志性有效成分的含量测定方法和指纹图谱检测方法,为穿破石的成分鉴别、质量评价以及质量标准的制定具有重要的意义。
发明内容
发明目的:本发明的目的是解决现有技术的不足,提供一种穿破石的标志性有效成分的含量测定方法和指纹图谱检测方法,该检测方法可以客观、全面、准确的评价穿破石的质量,对控制穿破石的质量和保证临床疗效具有重要意义。
技术方案:为了实现上述目的,本发明采取的技术方案为:
一种穿破石的质量检测方法,包括以下步骤:
步骤1,穿破石供试品溶液的制备:
取穿破石粉末,精密称定,置具塞锥形瓶中,精密加入甲醇50mL,称定重量,超声30min,放冷,滤过,滤液蒸干,用甲醇复溶,转移至10mL容量瓶,并用甲醇定容,用0.45μm微孔滤膜滤过,取续滤液,即得;
步骤2,混合对照品溶液的制备:
精密称取对照品白藜芦醇、花旗松素、槲皮素、桑色素、伞形花内酯、柚皮素、山奈酚、齐墩果酸、大黄素、大黄素甲醚、氧化白藜芦醇,加甲醇制得一定浓度的混合对照品溶液;
步骤3,标准曲线的建立:
取步骤2的混合对照品溶液依次稀释5倍,然后取10μL注入高效液相色谱仪,以对照品进样量X(μg)为横坐标,峰面积Y为纵坐标,绘制标准曲线方程;
步骤4,色谱图的测定:
分别精密吸取步骤1供试品溶液和步骤2混合对照品溶液各10μL,注入高效液相色谱仪,记录色谱图;
步骤5,指纹图谱的建立:
将步骤4中获得的穿破石供试品溶液的指纹图谱导出,并导入中药色谱指纹图谱相似度评价系统2012A;选择不同批次穿破石的色谱图中均存在的色谱峰作为共有峰;用平均值计算法生成穿破石的对照指纹图谱,计算各共有峰的相对保留时间和相对峰面积;并根据混合对照品溶液色谱图的保留时间标注对照指纹图谱中峰的化学成分。
作为优选方案,以上所述的穿破石的质量检测方法,步骤1穿破石供试品溶液制备方法为:精密称定基源为柘树类穿破石不同批次的样品1~5克,置具塞锥形瓶中,精密加入甲醇50mL,称定重量,超声30min,放冷,滤过,滤液蒸干,用甲醇复溶,转移至10mL容量瓶,并用甲醇定容,用0.45μm微孔滤膜滤过,取续滤液,即得基源为柘树类穿破石不同批次的供试品溶液。
作为优选方案,以上所述的穿破石的质量检测方法,步骤2混合对照品溶液的制备:分别精密称取对照品白藜芦醇、花旗松素、槲皮素、桑色素、伞形花内酯、柚皮素、山奈酚、齐墩果酸、大黄素、大黄素甲醚、氧化白藜芦醇,加甲醇制得浓度为0.0622mg/mL、0.2950mg/mL、0.0375mg/mL、0.1540mg/mL、0.1630mg/mL、0.1120mg/mL、0.0303mg/mL、0.0458mg/mL、0.0470mg/mL、1.1635m/mL的混合对照品溶液。
作为优选方案,以上所述的穿破石的质量检测方法,步骤3中,液相色谱条件为:YMC-PackODS-AC18色谱柱,规格为4.6mm×250mm,5μm;流动相为乙腈为A相-0.1%磷酸水为B相,梯度洗脱:0~10min,5%~5%A;10~20min,5%~10%A;20~30min,10%~10%A;30~50min,10%~20%A;50~60min,20%~20%A;60~90min,20%~35%A;90~120min,35%~55%A;120~170min,55%~55%A;170~195min,55%~90%A;195~200min,90%~5%A;200~205min,5%~5%A;;流速1mL/min;柱温30℃;进样量10μL;检测波长230nm、255nm、260nm、280nm。
作为优选方案,以上所述的穿破石的质量检测方法,对照品的标准曲线方程为:
花旗松素:y=36672x+139866,R=0.9998,柚皮素:y=32981x+18734,R=0.9999,山奈酚:y=37294x+211.21,R=0.9999,桑色素:y=41247x+19904,R=0.9999,槲皮素:y=44324x+1387.5,R=0.9998,大黄素:y=28020x+2737.9,R=0.9999,大黄素甲醚:y=12044x-10397,R=0.9999,白藜芦醇:y=42175x+10911,R=0.9999,伞形花内酯:y=15544x+13100,R=0.9999,氧化白藜芦醇:y=32981x+18734。
作为优选方案,所述的穿破石的质量检测方法,共鉴定18个指纹,保留时间分别为:1号峰28.237min,2号峰43.439min,3号峰53.76min,4号峰54.901min,5号峰73.787min,6号峰115.818min,7号峰121.981min,8号峰140.092min,9号峰153.089min,10号峰175.499min,11号峰185.882min,12号峰57.957min,13号峰88.053min,14号峰91.314min,15号峰80.693min,16号峰148.782min,17号峰46.848min,18号峰70.981min。
作为优选方案,所述的穿破石的质量检测方法,共鉴定18个指纹,其中3号峰为花旗松素,5号峰为桑色素,7号峰为大黄素,11号峰为齐墩果酸,12号峰为氧化白藜芦醇,13号峰为柚皮素,14号峰为山奈酚,15号峰为槲皮素,16号峰为大黄素甲醚,17号峰为伞形花内酯,18号峰为白藜芦醇。
一种穿破石的质量检测方法,还包括薄层色谱法鉴别:
(1)各取基源为柘树类穿破石药材粉末,精密称定,置具塞锥形瓶,精密加入甲醇,超声,放冷,滤过,滤液蒸干,甲醇复溶,转移至容量瓶,甲醇定容,即得基源为柘树类穿破石样品溶液;
(2)精密称取花旗松素、氧化白藜芦醇、桑色素、槲皮素、柚皮素、山奈酚,加甲醇制得混合对照品溶液;
(3)分别吸取步骤(1)基源为柘树类穿破石样品溶液和步骤(2)混合对照品溶液,分别点于同一块硅胶G薄层板上,以甲苯-乙酸乙酯-甲酸为展开剂,展开,取出,晾干,置紫外光灯检视;
在基源为柘树类穿破石供试品色谱中,具有与对照品花旗松素、氧化白藜芦醇、桑色素、槲皮素、柚皮素、山奈酚,相应的Rf位置上,显相同颜色的荧光斑点;
作为优选方案,以上所述的穿破石的质量检测方法,其特征在于,还包括薄层色谱法鉴别:
(1)各取基源为柘树类穿破石药材粉末,精密称定,置具塞锥形瓶,精密加入甲醇50mL,超声30min,放冷,滤过,滤液蒸干,甲醇复溶,转移至10mL容量瓶,甲醇定容,即得基源为柘树类穿破石类样品溶液;
(2)精密称取花旗松素、氧化白藜芦醇、桑色素、槲皮素、柚皮素、山奈酚加甲醇制得浓度为1.2028mg/mL、1.1733mg/mL、1.2360mg/mL、1.2493mg/mL、1.6435mg/mL、1.0114mg/mL的混合对照品溶液;
(3)分别吸取步骤(1)基源为柘树类穿破石样品溶液和步骤(2)混合对照品溶液各5~10μL,分别点于同一块硅胶G薄层板上,以体积比10:4:1的甲苯-乙酸乙酯-甲酸为展开剂,展开,取出,晾干,置365nm紫外光灯检视;
在基源为柘树类穿破石供试品色谱中,具有与对照品白藜芦醇、桑色素、槲皮素、柚皮素、山奈酚相应的Rf位置上,显相同颜色的荧光斑点。
有益效果:
1、本发明根据穿破石中所含的活性成分的结构性质特点,通过大量实验筛选出最佳的流动相组成,梯度洗脱程序、流速,检测波长、色谱柱,柱温等分析条件,经多次实验验证表明,本发明提供的穿破石指纹图谱检测方法,可以全面、客观、准确的检测和评价基源为柘树类穿破石的质量,为保证穿破石疗效具有重要意义。
2、用本发明所提供的方法所建立的穿破石指纹图谱,能有效地表征穿破石的质量,能客观体现各个构成指纹特征峰的前后顺序和相互关系,注重整体面貌特征,既可避免因测定个别化学成分而判定穿破石质量的片面性,又可减少为质量达标而人为处理的可能性。
附图说明
图1为基源为柘树类穿破石HPLC指纹图谱图。
图2为基源为柘树类穿破石样品4的HPLC色谱图。
图3为基源为柘树类穿破石样品和对照品薄层色谱图。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述,实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
以下实施例用到的仪器与试剂如下:
实验器材
1.1仪器
LC-20AT型高效液相色谱仪(SPD-M20A二极管阵列检测器)(日本岛津公司),XL-10B 500克密封型粉碎机(广州市旭朗机械设备有限公司),DHG-9140A电热恒温鼓风干燥箱(上海精宏实验设备有限公司),Linomat5半自动点样仪,CAMAG TLC-III型薄层扫描仪,KQ5200DE型数控超声波清洗器(昆山市超声仪器有限公司),HH-6数显恒温水浴锅(常州国华电器有限公司),SX2-4-10A型箱式电阻炉(郑州鑫涵仪器设备有限公司),SHZ-D(III)循环水式真空泵(巩义市予华仪器有限责任公司),BP211D型万分之一电子天平(北京赛多利斯仪器系统有限公司),FN1004N型十万分之一电子天平(北京赛多利斯仪器系统有限公司),硅胶板试剂(200×100mm,G板,青岛海洋化工有限公司),硅胶板试剂(100×100mm,G板,青岛海洋化工有限公司)。
1.2药物与试剂
氧化白藜芦醇对照品(批号1932,纯度98.1%)均购自上海诗丹德标准服务有限公司;白藜芦醇对照品(批号PS011367,纯度≥98.0%)、大黄素甲醚对照品(批号PS020626,纯度≥98.0%)、花旗松素对照品(批号PS012235,纯度≥98.0%)、大黄素对照品(批号PS010625,纯度≥99.0%)、槲皮素对照品(批号PS012093,纯度≥99.0%)、齐墩果酸对照品(批号PS012022,纯度≥98.5%)、桑色素对照品(批号PS012570,纯度≥98.0%)、伞形花内酯对照品(PS1412-0100,纯度≥98.0%)、柚皮素(批号PS011961,纯度≥98.0%)、山奈酚对照品(批号PS011676,纯度≥98.0%)均购自成都普思生物科技有限公司;乙腈为色谱纯,水为纯化水,其他试剂均为分析纯。穿破石柘树根对照药材(批号660029-201901)均购自中国食品药品检定研究院。所用穿破石样品为来源为市售。
8批次样品信息如下:
实施例1一种穿破石的质量检测方法,包括以下步骤:
步骤1,穿破石供试品溶液的制备:
分别精密称定基源为柘树类穿破石的不同批次的穿破石样品1克,置具塞锥形瓶中,精密加入甲醇50mL,称定重量,超声30min,放冷,滤过,滤液蒸干,用甲醇复溶,转移至10mL容量瓶,并用甲醇定容,用0.45μm微孔滤膜滤过,取续滤液,即得基源为柘树类穿破石不同批次的供试品溶液。
步骤2,混合对照品溶液的制备:
分别精密称取对照品白藜芦醇、花旗松素、槲皮素、桑色素、伞形花内酯、柚皮素、山奈酚、齐墩果酸、大黄素、大黄素甲醚、氧化白藜芦醇,加甲醇制得浓度为0.0622mg/mL、0.2950mg/mL、0.0375mg/mL、0.1540mg/mL、0.1630mg/mL、0.1120mg/mL、0.0303mg/mL、1.2086mg/mL、0.0458mg/mL、0.0470mg/mL、1.1635m/mL的混合对照品溶液;
步骤3,标准曲线的建立:
取步骤2的混合对照品溶液依次稀释5倍,然后取10μL注入高效液相色谱仪,以对照品进样量X(μg)为横坐标,峰面积Y为纵坐标,绘制标准曲线方程:各化合物标准曲线方程为:花旗松素:y=36672x+139866,R=0.9998,柚皮素:y=32981x+18734,R=0.9999,山奈酚:y=37294x+211.21,R=0.9999,桑色素:y=41247x+19904,R=0.9999,槲皮素:y=44324x+1387.5,R=0.9998,大黄素:y=28020x+2737.9,R=0.9999,大黄素甲醚:y=12044x-10397,R=0.9999,白藜芦醇:y=42175x+10911,R=0.9999,伞形花内酯:y=15544x+13100,R=0.9999。氧化白藜芦醇:y=32981x+18734。
步骤4,色谱图的测定:
分别精密吸取步骤1基源为柘树类穿破石供试品溶液和步骤2混合对照品溶液各10μL,注入高效液相色谱仪,记录色谱图;
液相色谱条件为:YMC-Pack ODS-A C18色谱柱,规格为4.6mm×250mm,5μm;流动相为乙腈为A相-0.1%磷酸水为B相,梯度洗脱:0~10min,5%~5%A;10~20min,5%~10%A;20~30min,10%~10%A;30~50min,10%~20%A;50~60min,20%~20%A;60~90min,20%~35%A;90~120min,35%~55%A;120~170min,55%~55%A;170~195min,55%~90%A;195~200min,90%~5%A;200~205min,5%~5%A;;流速1mL/min;柱温30℃;进样量10μL;检测波长230nm、255nm、260nm、280nm。
步骤5,指纹图谱的建立:
将步骤4中获得的基源为柘树类穿破石供试品溶液的指纹图谱导出,并导入中药色谱指纹图谱相似度评价系统2012A;选择不同批次穿破石的色谱图中均存在的色谱峰作为共有峰;用平均值计算法生成穿破石的对照指纹图谱如图1,样品4基源为柘树类穿破石供试品溶液的HPLC图谱见图2,计算各共有峰的相对保留时间和相对峰面积。共鉴定18个指纹,保留时间分别为:1号峰28.237min,2号峰43.439min,3号峰53.76min,4号峰54.901min,5号峰73.787min,6号峰115.818min,7号峰121.981min,8号峰140.092min,9号峰153.089min,10号峰175.499min,11号峰185.882min,12号峰57.957min,13号峰88.053min,14号峰91.314min,15号峰80.693min,16号峰148.782min,17号峰46.848min,18号峰70.981min。其中3号峰为花旗松素,5号峰为桑色素,7号峰为大黄素,11号峰为齐墩果酸,12号峰为氧化白藜芦醇,13号峰为柚皮素,14号峰为山奈酚,15号峰为槲皮素,16号峰为大黄素甲醚,17号峰为伞形花内酯,18号峰为白藜芦醇。
并通过计算不同批次的基源为柘树类穿破石(S1样品1,S2样品2,S3样品3,S4样品4,S5样品5,S6样品6,S7样品7,S8样品8)的相似度如下表1。
表1基源为柘树类穿破石指纹图谱相似度
并根据步骤3标准曲线方程计算基源为柘树类穿破石的不同批次样品中有效成分含量,如下表2。
表2基源为柘树类穿破石主要成分含量测定结果(mg/g)
步骤6,薄层色谱法鉴别:
(1)各取基源为柘树类穿破石药材粉末,精密称定,置具塞锥形瓶,精密加入甲醇50mL,超声30min,放冷,滤过,滤液蒸干,甲醇复溶,转移至10mL容量瓶,甲醇定容,即得基源为柘树类穿破石样品溶液;
(2)精密称取花旗松素、白藜芦醇、桑色素、槲皮素、柚皮素、山奈酚加甲醇制得浓度为1.2028mg/mL、1.1733mg/mL、1.2360mg/mL、1.2493mg/mL、1.6435mg/mL、1.0114mg/mL的混合对照品溶液;
(3)分别吸取步骤(1)基源为柘树类穿破石样品溶液和步骤(2)混合对照品溶液各5~10μL,分别点于同一块硅胶G薄层板上,以体积比10:4:1的甲苯-乙酸乙酯-甲酸为展开剂,展开,取出,晾干,置365nm紫外光灯检视;
在基源为柘树类穿破石供试品色谱中,具有与对照品花旗松素、氧化白藜芦醇、桑色素、槲皮素、柚皮素、山奈酚,相应的Rf位置上,显相同颜色的荧光斑点;薄层色谱图见图3。图3中基源为柘树类穿破石样品,C1-样品4,C2-样品8,C3-样品1,C4-样品6,C5-样品17,C6-样品20,C7-样品10,C8-样品19,C9-穿破石对照药材(柘树根);D1花旗松素,D2桑色素,D3柚皮素,D4山奈酚,D5槲皮素,D6氧化白藜芦醇。
实施例2指纹图谱检测的方法学研究
1、方法学考察
1、精密度试验
取4号样品,按按实施例1方法制备供试品溶液,按实施例1色谱条件连续进样6次,计算样4中各待测成分的保留时间和峰面积的RSD值,考察仪器的精密度。结果显示样品4中各待测成分的保留时间RSD值均小于1.5%,峰面积RSD值均小于3.0%。表明仪器的精密度良好。
2、重复性试验
分别取4号样品各6份,各约1g,精密称定,按实施例1方法制备供试品溶液,按实施例1下色谱条件依次进样测定,计算样品4中各待测成分含量的保留时间和含量的RSD值,考察方法的重复性。结果显示样品4中各待测成分的保留时间RSD值均小于1.5%,计算的含量的RSD值均小于3.0%,表明该方法的重复性良好。
3、稳定性试验
取4号样品,按实施例1方法制备供试品溶液,分别于制样后0,4,8,16,24h时按实施例1下色谱条件依次进样测定,计算样品4中各待测成分的保留时间和峰面积的RSD值,考察样品的稳定性。结果显示样品4中各待测成分的保留时间RSD值均小于1.5%,峰面积RSD值均小于3.0%。表明供试品溶液在制备24h内稳定。
4、加样回收率试验
分别取已知含量的样品粉末4号各3份,按其待测成分含量分别添加80%、100%、120%的混合对照品,按实施例1下色谱条件下进样,每份平行进样3针,计算样品4供试品中待测成分的平均加样回收率和RSD值,具体见表3。结果平均加样回收率在95%-105%之间,RSD值小于3%,表明准确性良好。
表3
以上实验结果表明,本发明提供的穿破石指纹图谱检测方法,稳定性好,精密度高,重复性好,能全面客观评价穿破石的质量,为保证临床疗效具有重要的意义。
以上实施例仅为本发明的示例性实施例,不用于限制本发明,本发明的保护范围由权利要求书限定。本领域技术人员可以在本发明的实质和保护范围内,对本发明做出各种修改或等同替换,这种修改或等同替换也应视为落在本发明的保护范围内。
Claims (6)
1.一种穿破石的质量检测方法,其特征在于,它包括指纹图谱检测和薄层色谱法鉴别;
所述的指纹图谱检测包括以下步骤:
步骤1、穿破石供试品溶液的制备:
取穿破石粉末,精密称定,置具塞锥形瓶中,精密加入甲醇50mL,称定重量,超声30min,放冷,滤过,滤液蒸干,用甲醇复溶,转移至10mL容量瓶,并用甲醇定容,用0.45μm微孔滤膜滤过,取续滤液,即得;
步骤2、混合对照品溶液的制备:
精密称取对照品白藜芦醇、花旗松素、槲皮素、桑色素、伞形花内酯、柚皮素、山奈酚、齐墩果酸、大黄素、大黄素甲醚、氧化白藜芦醇,加甲醇制得一定浓度的混合对照品溶液;
步骤3、标准曲线的建立:
取步骤2的混合对照品溶液依次稀释5倍,然后取10μL注入高效液相色谱仪,以对照品进样量Xμg为横坐标,峰面积Y为纵坐标,绘制标准曲线方程;
步骤4、色谱图的测定:
分别精密吸取步骤1供试品溶液和步骤2混合对照品溶液各10μL,注入高效液相色谱仪,记录色谱图;
步骤5、指纹图谱的建立:
将步骤4中获得的穿破石供试品溶液的液相色谱图谱导出,并导入中药色谱指纹图谱相似度评价系统2012A;选择不同批次穿破石的色谱图中均存在的色谱峰作为共有峰;用平均值计算法生成穿破石的对照指纹图谱,计算各共有峰的相对保留时间和相对峰面积;并根据混合对照品溶液色谱图的保留时间标注对照指纹图谱中峰的化学成分;
所述的步骤3中,液相色谱条件为:YMC-Pack ODS-A C18色谱柱,规格为4.6mm×250mm,5μm;流动相为乙腈为A相-0.1%磷酸水为B相,梯度洗脱:0~10min,5%~5%A;10~20min,5%~10%A;20~30min,10%~10%A;30~50min,10%~20%A;50~60min,20%~20%A;60~90min,20%~35%A;90~120min,35%~55%A;120~170min,55%~55%A;170~195min,55%~90%A;195~200min,90%~5%A;200~205min,5%~5%A;流速1mL/min;柱温30℃;进样量10μL;检测波长230nm、255nm、260nm、280nm;
所述的薄层色谱法鉴别包括以下步骤:
(1)各取基源为柘树类穿破石药材粉末,精密称定,置具塞锥形瓶,精密加入甲醇50mL,超声30min,放冷,滤过,滤液蒸干,甲醇复溶,转移至10mL容量瓶,甲醇定容,即得基源为柘树类穿破石样品溶液;
(2)精密称取花旗松素、氧化白藜芦醇、桑色素、槲皮素、柚皮素、山奈酚加甲醇制得浓度为1.2028mg/mL、1.1733mg/mL、1.2360mg/mL、1.2493mg/mL、1.6435mg/mL、1.0114mg/mL的混合对照品溶液;
(3)分别吸取步骤(1)基源为柘树类穿破石样品溶液和步骤(2)混合对照品溶液各5~10μL,分别点于同一块硅胶G薄层板上,以体积比10:4:1的甲苯-乙酸乙酯-甲酸为展开剂,展开,取出,晾干,置365nm紫外光灯检视;
在基源为柘树类穿破石供试品色谱中,具有与对照品花旗松素、氧化白藜芦醇、桑色素、槲皮素、柚皮素、山奈酚相应的Rf位置上,显相同颜色的荧光斑点。
2.根据权利要求1所述的穿破石的质量检测方法,其特征在于,步骤1穿破石供试品溶液制备方法为:精密称定基源为柘树类穿破石不同批次的样品1~5克,置具塞锥形瓶中,精密加入甲醇50mL,称定重量,超声30min,放冷,滤过,滤液蒸干,用甲醇复溶,转移至10mL容量瓶,并用甲醇定容,用0.45μm微孔滤膜滤过,取续滤液,即得基源为柘树类穿破石不同批次的供试品溶液。
3.根据权利要求1所述的穿破石的质量检测方法,其特征在于,步骤2混合对照品溶液的制备:分别精密称取对照品白藜芦醇、花旗松素、槲皮素、桑色素、伞形花内酯、柚皮素、山奈酚、大黄素、大黄素甲醚、氧化白藜芦醇,加甲醇制得浓度为0.0622mg/mL、0.2950mg/mL、0.0375mg/mL、0.1540mg/mL、0.1630mg/mL、0.1120mg/mL、0.0303mg/mL、0.0458mg/mL、0.0470mg/mL、1.1635m/mL的混合对照品溶液。
4.根据权利要求1至3任一项所述的穿破石的质量检测方法,其特征在于,对照品的标准曲线方程为:
花旗松素:y=36672x+139866,R=0.9998,柚皮素:y=32981x+18734,R=0.9999,山奈酚:y=37294x+211.21,R=0.9999,桑色素:y=41247x+19904,R=0.9999,槲皮素:y=44324x+1387.5,R=0.9998,大黄素:y=28020x+2737.9,R=0.9999,大黄素甲醚:y=12044x-10397,R=0.9999,白藜芦醇:y=42175x+10911,R=0.9999,伞形花内酯:y=15544x+13100,R=0.9999、氧化白藜芦醇:y=32981x+18734。
5.根据权利要求1所述的穿破石的质量检测方法,其特征在于,共鉴定18个指纹,保留时间分别为:1号峰28.237min,2号峰43.439min,3号峰53.76min,4号峰54.901min,5号峰73.787min,6号峰115.818min,7号峰121.981min,8号峰140.092min,9号峰153.089min,10号峰175.499min,11号峰185.882min,12号峰57.957min,13号峰88.053min,14号峰91.314min,15号峰80.693min,16号峰148.782min,17号峰46.848min,18号峰70.981min。
6.根据权利要求1所述的穿破石的质量检测方法,其特征在于,共鉴定18个指纹,3号峰为花旗松素,5号峰为桑色素,7号峰为大黄素,11号峰为齐墩果酸,12号峰为氧化白藜芦醇,13号峰为柚皮素,14号峰为山奈酚,15号峰为槲皮素,16号峰为大黄素甲醚,17号峰为伞形花内酯,18号峰为白藜芦醇。
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