Establishment of characteristic chromatograms of TLC and HPLC and determination of six components in water

目的 建立旋覆花水提物的薄层色谱（TLC）和高效液相色谱（HPLC）的特征图谱,同时建立水提物中6种成分（绿原酸、咖啡酸、异槲皮苷、1,5-二咖啡酰奎宁酸、4,5-二咖啡酰奎宁酸和旋覆花内酯）的含量测定方法,评价11批旋覆花药材水提物的相似性。方法 （1）采用HF254型薄层层析硅胶板、正丁醇-丙酮-吡啶-水-冰醋酸（1∶5∶3∶1∶10 μL）为展开剂,在365 nm波长下检视喷洒10%硫酸-乙醇前、后的色谱斑点,建立TLC特征图谱。（2）采用SinoChrom ODS-BP（4.6 mm×250 mm,5 μm）色谱柱,流动相为0.05%磷酸溶液-乙腈的梯度洗脱程序,流速0.8 mL/min,柱温35℃,检测波长205 nm,建立HPLC特征图谱。（3）6种成分的含量测定条件与（2）相同,但检测波长分别为203 nm（旋覆花内酯）、256 nm（异槲皮苷）和327 nm（咖啡酸、绿原酸、1,5-二咖啡酰奎宁酸、4,5-二咖啡酰奎宁酸）。结果 （1）喷洒显色剂前、后的TLC图谱均显示出良好的特征性。（2）HPLC特征图谱显示含有28个共有峰,指认出10个峰（绿原酸、咖啡酸、1,5-二咖啡酰奎宁酸、4,5-二咖啡酰奎宁酸、花旗松素、芦丁、槲皮苷、异槲皮苷、木犀草素和旋覆花内酯）。通过相似度评价和聚类分析可知11批旋覆花水提物相似度良好。（3）绿原酸、咖啡酸、异槲皮苷、1,5-二咖啡酰奎宁酸、4,5-二咖啡酰奎宁酸和旋覆花内酯6种成分的线性范围分别为3.25~65.00、4.00~80.00、1.60~32.00、17.75~355.00、2.50~50.00和3.75~75.00 μg/mL。11批旋覆花水提物中绿原酸、咖啡酸、异槲皮苷、1,5-二咖啡酰奎宁酸、4,5-二咖啡酰奎宁酸和旋覆花内酯的平均含量分别为3.38~6.66、2.13~7.35、0.51~1.57、15.81~24.87、0.50~1.08和0.77~4.73 μg/mg。结论 该方法的TLC和HPLC特征图谱分离效果良好,含量测定方法可靠,可为旋覆花水提物质量控制方法提供实验依据。

Abstract

Objective To establish the characteristic chromatograms of thin-layer chromatography （TLC） and high-performance liquid chromatography （HPLC） of the aqueous-extract of Inula japonica Thunb.,and to determine the contents of six components （chlorogenic acid,caffeic acid,isoquercitrin,1,5-dicaffeoylquinic acid,4,5-dicaffeoylquinic acid and inulicin） in the aqueous-extract. Methods （1）The HF254 TLC silica gel plate,developing solvents of n-butanol-acetone-pyridine-water-glacial acetic acid （1∶5∶3∶1∶10 μL） and 10% sulphuric acid-ethanol were used before the chromatographic spots were examined at 365 nm. （2）The Sino Chrom ODS-BP column （ϕ4.6 mm× 250 mm,5 μm） and a mobile phase of 0.05% phosphoric acid solution and acetonitrile were used. The flow rate was 0.8 mL/min,the column temperature 35℃ and detection wavelength 205 nm. （3）The analytic conditions of the six components were similar to those described in method （2）,but the wavelengths were respectively 203 nm （inulicin）,256 nm （isoquercitrin） and 327 nm （caffeic acid,chlorogenic acid,1,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid）. Results （1）The TLC chromatograms showed fine properties before and after the colour developer was sprayed. （2）Twenty-eight common peaks were marked in the HPLC characteristic chromatogram,so were ten components （chlorogenic acid,caffeic acid,1,5-dicaffeoylquinic acid,4,5-dicaffeoylquinic acid,taxifolin, rutin, quercitrin, isoquercitrin, luteolin and inulicin）. The aqueous-extracts of eleven batches of materials of Inula japonica shared much similarity based on similarity and cluster analysis. （3） There were good linear relationships,and the linear ranges of chlorogenic acid,caffeic acid,isoquercitrin,1,5-dicaffeoylquinic acid,4,5-dicaffeoylquinic acid and inulicin were 3.25-65.00 μg/mL,4.00-80.00 μg/mL,1.60-32.00 μg/mL,17.75-355.00 μg/mL,2.50-50.00 μg/mL and 3.75-75.00 μg/mL,respectively. The contents of chlorogenic acid,caffeic acid,isoquercitrin,1,5-dicaffeoylquinic acid,4,5-dicaffeoylquinic acid and inulicin in elven batches of water-extract from Inula japonica were 3.38-6.66 μg/mg,2.13-7.35 μg/mg,0.51-1.57 μg/mg,15.81-24.87 μg/mg,0.50-1.08 μg/mg and 0.77-4.73 μg/mg,respectively. Conclusion The specific TLC and HPLC characteristic chromatograms can help ensure accurate quantitative determination of six components of the water-extract of Inula japonica,which can be used for quality control of the water-extract of Inula japonica.

关键词

旋覆花 /水提物 /薄层色谱 /高效液相色谱 /特征图谱 /定量分析{{custom_keyword}} /

Key words

Inula japonica /aqueous-extract /thin-layer chromatography /high-performance liquid chromatography /characteristic chromatogram /quantitative analysis{{custom_keyword}} /

孟凡恒, 骆芙瑶, 冯婷, 杨郁, 徐锐, 朱帅鸣, 单俊杰.旋覆花水提物的TLC和HPLC特征图谱及6种化学成分的含量测定. 军事医学. 2024, 48(3): 202-212 https://doi.org/10.7644/j.issn.1674-9960.2024.03.007

MENG Fanheng, LUO Fuyao, FENG Ting, YANG Yu, XU Rui, ZHU Shuaiming, SHAN Junjie.Establishment of characteristic chromatograms of TLC and HPLC and determination of six components in water-extract of Inula japonica. Military Medical Sciences. 2024, 48(3): 202-212 https://doi.org/10.7644/j.issn.1674-9960.2024.03.007

参考文献

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