目的 探讨10种不同产地规格浙贝母超高效液相色谱-四极杆-飞行时间质谱(UPLC-Q-TOF-MS)指纹谱与其化痰作用的谱-效关系,初步筛选浙贝母化痰作用质量标志物(Q-marker),并比较主要质量标志物的含量差异。方法 应用UPLC-Q-TOF-MS技术建立浙贝母指纹图谱。液相条件:色谱柱ZORBAX HPLC Eclipse Plus C18(3 mm×150 mm,1.8 μm),以乙腈-0.1% 甲酸水为流动相进行梯度洗脱,流速0.4 mL·min-1;柱温30 ℃;进样量2 μL。质谱条件:通过运用电喷雾(ESI)离子源,在正负离子模式下进行数据采集。采用小鼠酚红法以气管酚红排泌量为指标评价浙贝母化痰作用。结合双变量相关分析和灰色关联度分析研究浙贝母指纹图谱中共有峰与化痰作用的相关性,筛选出关联性较大的成分作为其化痰作用的主要质量标志物。采用高效液相色谱-蒸发光散射检测(HPLC-ELSD)建立其中4个主要成分的含量测定方法,并比较不同产地规格的含量差异。结果 初步筛选出6个与浙贝母化痰作用相关的质量标志物,分别为腺苷、贝母辛,西贝素、贝母素乙、贝母素甲、浙贝素。10个不同产地规格浙贝母中贝母辛含量在0.186~1.106 9 mg·g-1内;西贝素含量在0.204 7~1.243 9 mg·g-1内;贝母素乙含量在0.084 2~0.611 5 mg·g-1内;贝母素甲含量在1.458 5~11.769 6 mg·g-1内。结论 基于“谱-效”关联的质量标志物研究策略是一种快速分析及初步确定浙贝母化痰作用药效物质的有效方法。在此基础上建立的浙贝母主要质量标志物HPLC-ELSD含量测定的方法可靠、准确。本实验对完善浙贝母质量控制方法,制定科学的浙贝母商品标准规范具有较重要的参考价值。
Abstract
OBJECTIVE To explore the relationship between the UPLC-Q-TOF-MS fingerprints of Fritillaria thunbergii and its phlegm-reducing effect in 10 different producing areas, screen the quality markers of phlegm-removing phlegm of Fritillaria thunbergii, and compare the content of main components. METHODS UPLC-Q-TOF-MS technology was used to establish the fingerprint of Fritillaria thunbergii. Liquid conditions: chromatographic column ZORBAXHPLC Eclipse Plus C18(3 mm×150 mm, 1.8 μm), gradient elution with methanol-0.1% formic acid water as mobile phase, flow rate was 0.4 mL·min-1; column temperature was maintained at 30 ℃; injection volume was 2 μL. Mass spectrometry conditions: an electrospray (ESI) ion source was used to collect data in positive and negative ion mode. The mouse phenol red method was used to evaluate the phlegm-reducing effect of Fritillaria thunbergii with tracheal phenol red excretion. Combining bivariate correlation analysis and gray correlation analysis to study the correlation between the common peak in the fingerprint of Fritillaria thunbergii and the phlegm effect, and screen out the components with greater correlation as quality markers for its phlegm effect, and used HPLC-ELSD method established the determination method for the content of 4 main components. RESULTS Six quality markers related to the phlegm-reducing effect of Fritillaria thunbergii were screened out: adenosine, peimisine, imperialine, peiminine, peimine, and zhebeine. The content of peimisine was in the range of 0.186-1.106 9 mg·g-1; the content of imperialine was in the range of 0.204 7-1.243 9 mg·g-1; the content of peiminine was in the range of 0.084 2-0.611 5 mg·g-1; the content of peimin was in 1.458 5-11.769 6 mg·g-1. CONCLUSION The research strategy of quality markers based on "spectrum-effect" correlation is an effective method for rapid analysis and preliminary determination of the effective components of Fritillaria thunbergii Miq. in resolving phlegm.On this basis, the established HPLC-ELSD method for determination of the main quality marker of Fritillaria thunbergii is reliable and accurate.This study has important reference value for improving the quality control method and making scientific commodity standards with Fritillaria thunbergii Miq.
关键词
浙贝母 /
谱效关系 /
超高效液相色谱-四极杆-飞行时间质谱 /
高效液相色谱-蒸发光散射检测 /
质量标志物
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Key words
Fritillaria thunbergii /
spectrum effect relationship /
UPLC-Q-TOF-MS /
HPLC-ELSD /
quality marker
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中图分类号：
R917
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脚注
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基金
浙江省公益性技术应用研究计划资助(LGN19H280002);浙江省食品药品监督管理局2018年度科技计划项目资助(2018018);宁波市科技创新2025重大专项资助(2019B10008); 2020年度高等学校国内访问工程师“校企合作项目”资助(FG2020003)
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