中药肿节风质量控制方法研究
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摘要
肿节风是金粟兰科植物草珊瑚(Sarcandra glabra (Thunb.) Nakai)的干燥全株,具有抗菌、消炎和抗肿瘤等作用。目前全国有56家药厂以肿节风为原料生产相关制剂。中药肿节风及相关产品的质量控制存在着质量标准低、检测指标和方法混乱、缺乏对产品生产中问环节的肿节风水提物的严格质量监控、不能反映抗炎活性等诸多问题,因而无法进行全面、有效、稳定的整体质量控制,亟待开展进一步的研究。
本文以课题组前期对肿节风水提物的化学成分及其活性研究结果为基础,从肿节风生产过程的中间环节入手,针对中药肿节风水提物开展指纹图谱研究并对主要色谱峰进行明确化学指认、建立包含指标成分及抗炎活性成分的多成分含量测定方法及采用高效液相色谱-电喷雾多级质谱联用技术(HPLC-ESI-MSn)进行部分化学成分的快速检识,为提升肿节风水提物及制剂的质量控制方法提供科学依据,并为肿节风水提物的国家药品标准的制订提供数据支持。
一、肿节风水提物及其制剂的HPLC指纹图谱研究
本章旨在建立肿节风水提物的HPLC指纹图谱并对其主要色谱峰进行明确的化学指认,对肿节风水提物及其市售制剂的质量进行评价。该方法采用UltimateXB-C18色谱柱,以乙腈-水(各含0.1%甲酸)为流动相进行梯度洗脱,流速为1.0mL／min,柱温为30℃,检测波长为330 nm,测定肿节风水提物的HPLC指纹图谱,并以共有模式的对照指纹图谱为对照,对不同厂家生产的12批肿节风水提物和20批市售制剂进行相似度评价。结果表明,建立的肿节风水提物的HPLC指纹图谱具有良好的稳定性、精密度和重复性,不同厂家生产的12批肿节风水提物和20批市售制剂与共有模式对照指纹图谱的相似度分别在0.883～0.987和0.797～0.982问；其中8个主要色谱峰经化学指认,分别为新绿原酸、绿原酸、隐绿原酸、β-hydroxypropiovanillone、咖啡酸、异嗪皮啶、迷迭香酸-4-O-葡萄糖苷和迷迭香酸；本研究所建立的HPLC指纹图谱涵盖了肿节风水提物中的抗炎活性成分与指标成分,反映了肿节风水提物及相关制剂的多成分的整体面貌,为全面、有效控制肿节风及相关制剂的质量奠定了科学基础。
二、肿节风水提物及其制剂中迷迭香酸和异嗪皮啶含量的HPLC-UVD法测定
本章旨在建立肿节风水提物中包含抗炎活性成分及指标成分的多成分含量测定方法。该方法采用Ultimate XB-C18色谱柱(250 mm×4.6 1mm,5μm),以乙腈-水(各含0.1%的甲酸)为流动相进行梯度洗脱,流速为1.00 mL／min,柱温为35℃,检测波长为330 nm。迷迭香酸在0.04746～1.898μg和异嗪皮啶在0.01018～0.4072μg呈现良好线性关系；仪器精密度试验、重复性试验、稳定性试验的RSD均不大于2.0%；迷迭香酸与异嗪皮啶在肿节风水提取物中的回收率分别为99.8%和99.2%,RSD分别为0.70%和0.63%(n=6)；而在相关制剂中的回收率分别为100.7%和98.4%,RSD分别为1.6%和0.44%(n=6)；肿节风水提取物中迷迭香酸的含量在1.479～6.447 mg／g间、异嗪皮啶的含量在1.912～3.540 mg／g间；在相关制剂中,迷迭香酸的含量在0.847～15.946 mg/g间、异嗪皮啶的含量在2.032～4.093 mg／g间。结果表明,本研究所建立的含量测定方法简便、快速、专属性强、准确度、精密度和稳定性良好,可为建立反映肿节风水提物抗炎活性的多成分含量测定方法和提升肿节风水提物及相关制剂的质量标准提供科学依据。
三、肿节风水提物中咖啡酰类衍生物的HPLC-ESI-MSn快速检识
本章旨在采用高效液相色谱-电喷雾-多级质谱联用技术(HPLC-ESI-MSn)对中药肿节风水提物中可能的抗炎活性物质群——咖啡酰类衍生物进行快速检识研究。通过文献调研和对经分离得到的绿原酸、新绿原酸,隐绿原酸、咖啡酸,迷迭香酸和迷迭香酸4-O-β-D葡萄糖苷等化学对照品的电喷雾-多级质谱裂解行为和紫外DAD吸收光谱图的分析,总结出咖啡酰类多聚体化合物存在198,180Da的特征中性丢失,结合相应的碎片离子如m／z197,m／z179,m／z 135可判断母核中含有一个丹参素结构片段,而利用Aλmax=325±5nm和Aλmax=325±5 mn／Aλmax=280±5nm可对咖啡酰多聚体的聚合单元和聚合位点作初步判断。利用上述规律,本研究采用HPLC-ESI／MSn技术,从肿节风水提物在负离子模式下的一级全扫描总离子流质量色谱图中的45个色谱峰中,快速检识了19个色谱峰为咖啡酰类衍生物,对其中6个根据对照品进行了明确指认,并对13个可能的结构进行了推测。研究结果有助于进一步了解中药肿节风中咖啡酰类衍生物、阐明肿节风抗炎作用物质基础,从而为更为有效地评价及控制中药肿节风的质量提供一定参考。
Hebra Sarcandrae was the whole plant of Sarcandra glabra (Thunb.) Nakai, which was used as antibiotic, anti-inflammatory and anti-tumor traditional Chinese medicine (TCM).Up to now, there were 56 domestic manufactures for Hebra Sarcandrae products, with low quality standards and poor quality control methods which are independent of anti-imflammatory activity and whole process quality control. Thus a further intensive study was needed urgently to improve the quality control methods of Hebra Sarcandrae and its products.
On the basis of our research on chemical constituents and their bioactivities in Hebra Sarcandrae water extract which was an important intermediate step in manufacturing process of its preparations, three parts of research were carried out in this thesis for improving the quality control of Hebra Sarcandrae water extract and its preparations as well as formulation of the national standard of Hebra Sarcandrae water extract.
1. Study on HPLC fingerprint chromatogram of Hebra Sarcandrae water extract and its preparations
This section mainly focused on establishing an HPLC fingerprint chromatogram of Hebra Sarcandrae water extract and its preparations with the main peaks being chemically identified. The method was developed on a C18 column (250 mm×4.6 mm i.d.,5μm) by gradient elution with acetonitrile-water (both containing 0.1% formic acid) as mobile phase at a flow rate of 1.00 mL/min with the wavelength at 330 nm. As a result the contrast HPLC fingerprint chromatogram was set up with good stability, precision and reproducibility by statistical and similarity analysis, then it was used as reference to evaluate the quality of the water extract and its preparations via similarity assay. The similarity of fingerprint chromatograms for 12 batches of Hebra Sarcandrae extract and 20 batches of its preparations ranged in 0.883～0.987 and 0.797～0.982 respectively. Furthermore, eight peaks in the chromatogram were identified as neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid,β-hydroxypropiovanillone, caffeic acid, isofraxidin, rosmarinic acid-4-O-glucoside, rosmarinic acid, respectively. Our investigation on HPLC fingerprint chromatogram with anti-inflammatory and characteristic components included, which reflected the profile of multi-component in Hebra Sarcandrae water extract and its preparations, would contribute to its whole process and effective quality control.
2. Assay of rosmarinic acid and isofraxidinin in Hebra Sarcandrae water extract and preparations by HPLC-UVD
This section mainly focused on developing an HPLC-UVD method for assay of multi-component including rosmarinic acid as anti-inflammatory component and isofraxidin as characteristic component in Sarcandra water extract and its preparations. The method was developed on a C18 column (250 mm×4.6 mm,5μm) by gradient elution with acetonitrile-water (both containing 0.1% formic acid) as mobile phase at a flow rate of 1.00 mL/min with the wavelength at 330 nm and column temperature at 35℃. The linearity was over the range of 0.04746～1.898μg for rosmarinic acid and 0.01018～0.4072μg for isofraxidin; The RSD was no more than 2.0% in precision test, reproducibility test and stability test, respectively; The average recovery of rosmarinic acid and isofraxidin in Hebra Sarcandrae extract were 99.8%(RSD=0.70%) and 99.2% (RSD=0.63%), while in paparations were 100.7% (RSD=1.6%) and 98.4% (RSD=0.44%), respectively. The twelve batches of Hebra Sarcandrae water extract sample shared an average content over the range of 1.479-6.447 mg/g for rosmarinic acid and 1.912-3.540 mg/g for isofraxidin, while 0.847-15.946 mg/g for rosmarinic acid and 2.032-4.093 mg/g for isofraxidin in its preparations. The method was simple, specific, precise and accurate and would help to improve the quality standard of Hebra Sarcandrae water extract and its preparations.
3. Investigation on rapid recognition of caffeic acid derivatives in Hebra Sarcandrae water extract by HPLC-ESI-MS"
This section mainly focused on rapid recognition of caffeic acid derivatives which were potential anti-inflammatory activity components in Hebra Sarcandrae water extract by HPLC-ESI-MSn. Based on the features of MS" fragmentation ions and DAD absorption spectra of caffeic acid derivatives in literature incorporated with those of reference chemical substances isolated from Hebra Sarcandrae water extract, a total of 19 caffeic acid derivatives out of 45 peaks in ESI(-)TIC MS chromatogram were rapidly recognized by HPLC-ESI-MSn,6 of them were identified compared with reference chemical substances, and the others were tentatively recognized according to the features of MSn fragmentation ions and DAD absorption spectra. This result would contribute to illuminate the relationship between anti-inflammatory activity and the related component in Hebra Sarcandrae.
本文以课题组前期对肿节风水提物的化学成分及其活性研究结果为基础,从肿节风生产过程的中间环节入手,针对中药肿节风水提物开展指纹图谱研究并对主要色谱峰进行明确化学指认、建立包含指标成分及抗炎活性成分的多成分含量测定方法及采用高效液相色谱-电喷雾多级质谱联用技术(HPLC-ESI-MSn)进行部分化学成分的快速检识,为提升肿节风水提物及制剂的质量控制方法提供科学依据,并为肿节风水提物的国家药品标准的制订提供数据支持。
一、肿节风水提物及其制剂的HPLC指纹图谱研究
本章旨在建立肿节风水提物的HPLC指纹图谱并对其主要色谱峰进行明确的化学指认,对肿节风水提物及其市售制剂的质量进行评价。该方法采用UltimateXB-C18色谱柱,以乙腈-水(各含0.1%甲酸)为流动相进行梯度洗脱,流速为1.0mL／min,柱温为30℃,检测波长为330 nm,测定肿节风水提物的HPLC指纹图谱,并以共有模式的对照指纹图谱为对照,对不同厂家生产的12批肿节风水提物和20批市售制剂进行相似度评价。结果表明,建立的肿节风水提物的HPLC指纹图谱具有良好的稳定性、精密度和重复性,不同厂家生产的12批肿节风水提物和20批市售制剂与共有模式对照指纹图谱的相似度分别在0.883～0.987和0.797～0.982问；其中8个主要色谱峰经化学指认,分别为新绿原酸、绿原酸、隐绿原酸、β-hydroxypropiovanillone、咖啡酸、异嗪皮啶、迷迭香酸-4-O-葡萄糖苷和迷迭香酸；本研究所建立的HPLC指纹图谱涵盖了肿节风水提物中的抗炎活性成分与指标成分,反映了肿节风水提物及相关制剂的多成分的整体面貌,为全面、有效控制肿节风及相关制剂的质量奠定了科学基础。
二、肿节风水提物及其制剂中迷迭香酸和异嗪皮啶含量的HPLC-UVD法测定
本章旨在建立肿节风水提物中包含抗炎活性成分及指标成分的多成分含量测定方法。该方法采用Ultimate XB-C18色谱柱(250 mm×4.6 1mm,5μm),以乙腈-水(各含0.1%的甲酸)为流动相进行梯度洗脱,流速为1.00 mL／min,柱温为35℃,检测波长为330 nm。迷迭香酸在0.04746～1.898μg和异嗪皮啶在0.01018～0.4072μg呈现良好线性关系；仪器精密度试验、重复性试验、稳定性试验的RSD均不大于2.0%；迷迭香酸与异嗪皮啶在肿节风水提取物中的回收率分别为99.8%和99.2%,RSD分别为0.70%和0.63%(n=6)；而在相关制剂中的回收率分别为100.7%和98.4%,RSD分别为1.6%和0.44%(n=6)；肿节风水提取物中迷迭香酸的含量在1.479～6.447 mg／g间、异嗪皮啶的含量在1.912～3.540 mg／g间；在相关制剂中,迷迭香酸的含量在0.847～15.946 mg/g间、异嗪皮啶的含量在2.032～4.093 mg／g间。结果表明,本研究所建立的含量测定方法简便、快速、专属性强、准确度、精密度和稳定性良好,可为建立反映肿节风水提物抗炎活性的多成分含量测定方法和提升肿节风水提物及相关制剂的质量标准提供科学依据。
三、肿节风水提物中咖啡酰类衍生物的HPLC-ESI-MSn快速检识
本章旨在采用高效液相色谱-电喷雾-多级质谱联用技术(HPLC-ESI-MSn)对中药肿节风水提物中可能的抗炎活性物质群——咖啡酰类衍生物进行快速检识研究。通过文献调研和对经分离得到的绿原酸、新绿原酸,隐绿原酸、咖啡酸,迷迭香酸和迷迭香酸4-O-β-D葡萄糖苷等化学对照品的电喷雾-多级质谱裂解行为和紫外DAD吸收光谱图的分析,总结出咖啡酰类多聚体化合物存在198,180Da的特征中性丢失,结合相应的碎片离子如m／z197,m／z179,m／z 135可判断母核中含有一个丹参素结构片段,而利用Aλmax=325±5nm和Aλmax=325±5 mn／Aλmax=280±5nm可对咖啡酰多聚体的聚合单元和聚合位点作初步判断。利用上述规律,本研究采用HPLC-ESI／MSn技术,从肿节风水提物在负离子模式下的一级全扫描总离子流质量色谱图中的45个色谱峰中,快速检识了19个色谱峰为咖啡酰类衍生物,对其中6个根据对照品进行了明确指认,并对13个可能的结构进行了推测。研究结果有助于进一步了解中药肿节风中咖啡酰类衍生物、阐明肿节风抗炎作用物质基础,从而为更为有效地评价及控制中药肿节风的质量提供一定参考。
Hebra Sarcandrae was the whole plant of Sarcandra glabra (Thunb.) Nakai, which was used as antibiotic, anti-inflammatory and anti-tumor traditional Chinese medicine (TCM).Up to now, there were 56 domestic manufactures for Hebra Sarcandrae products, with low quality standards and poor quality control methods which are independent of anti-imflammatory activity and whole process quality control. Thus a further intensive study was needed urgently to improve the quality control methods of Hebra Sarcandrae and its products.
On the basis of our research on chemical constituents and their bioactivities in Hebra Sarcandrae water extract which was an important intermediate step in manufacturing process of its preparations, three parts of research were carried out in this thesis for improving the quality control of Hebra Sarcandrae water extract and its preparations as well as formulation of the national standard of Hebra Sarcandrae water extract.
1. Study on HPLC fingerprint chromatogram of Hebra Sarcandrae water extract and its preparations
This section mainly focused on establishing an HPLC fingerprint chromatogram of Hebra Sarcandrae water extract and its preparations with the main peaks being chemically identified. The method was developed on a C18 column (250 mm×4.6 mm i.d.,5μm) by gradient elution with acetonitrile-water (both containing 0.1% formic acid) as mobile phase at a flow rate of 1.00 mL/min with the wavelength at 330 nm. As a result the contrast HPLC fingerprint chromatogram was set up with good stability, precision and reproducibility by statistical and similarity analysis, then it was used as reference to evaluate the quality of the water extract and its preparations via similarity assay. The similarity of fingerprint chromatograms for 12 batches of Hebra Sarcandrae extract and 20 batches of its preparations ranged in 0.883～0.987 and 0.797～0.982 respectively. Furthermore, eight peaks in the chromatogram were identified as neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid,β-hydroxypropiovanillone, caffeic acid, isofraxidin, rosmarinic acid-4-O-glucoside, rosmarinic acid, respectively. Our investigation on HPLC fingerprint chromatogram with anti-inflammatory and characteristic components included, which reflected the profile of multi-component in Hebra Sarcandrae water extract and its preparations, would contribute to its whole process and effective quality control.
2. Assay of rosmarinic acid and isofraxidinin in Hebra Sarcandrae water extract and preparations by HPLC-UVD
This section mainly focused on developing an HPLC-UVD method for assay of multi-component including rosmarinic acid as anti-inflammatory component and isofraxidin as characteristic component in Sarcandra water extract and its preparations. The method was developed on a C18 column (250 mm×4.6 mm,5μm) by gradient elution with acetonitrile-water (both containing 0.1% formic acid) as mobile phase at a flow rate of 1.00 mL/min with the wavelength at 330 nm and column temperature at 35℃. The linearity was over the range of 0.04746～1.898μg for rosmarinic acid and 0.01018～0.4072μg for isofraxidin; The RSD was no more than 2.0% in precision test, reproducibility test and stability test, respectively; The average recovery of rosmarinic acid and isofraxidin in Hebra Sarcandrae extract were 99.8%(RSD=0.70%) and 99.2% (RSD=0.63%), while in paparations were 100.7% (RSD=1.6%) and 98.4% (RSD=0.44%), respectively. The twelve batches of Hebra Sarcandrae water extract sample shared an average content over the range of 1.479-6.447 mg/g for rosmarinic acid and 1.912-3.540 mg/g for isofraxidin, while 0.847-15.946 mg/g for rosmarinic acid and 2.032-4.093 mg/g for isofraxidin in its preparations. The method was simple, specific, precise and accurate and would help to improve the quality standard of Hebra Sarcandrae water extract and its preparations.
3. Investigation on rapid recognition of caffeic acid derivatives in Hebra Sarcandrae water extract by HPLC-ESI-MS"
This section mainly focused on rapid recognition of caffeic acid derivatives which were potential anti-inflammatory activity components in Hebra Sarcandrae water extract by HPLC-ESI-MSn. Based on the features of MS" fragmentation ions and DAD absorption spectra of caffeic acid derivatives in literature incorporated with those of reference chemical substances isolated from Hebra Sarcandrae water extract, a total of 19 caffeic acid derivatives out of 45 peaks in ESI(-)TIC MS chromatogram were rapidly recognized by HPLC-ESI-MSn,6 of them were identified compared with reference chemical substances, and the others were tentatively recognized according to the features of MSn fragmentation ions and DAD absorption spectra. This result would contribute to illuminate the relationship between anti-inflammatory activity and the related component in Hebra Sarcandrae.
引文
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