目的:建立同时测定黄芪中毛蕊异黄酮葡萄糖苷、刺芒柄花苷、毛蕊异黄酮、山柰酚、异鼠李素和芒柄花素含量的超高效液相方法,并对结果数据进行初步分析。方法:使用Phenomenex Luna Omega C18色谱柱(100 mm×2.1 mm,1.6 μm),以0.1%甲酸水溶液-甲醇为流动相,进行梯度洗脱,流速0.4 mL·min-1,柱温35 ℃,检测波长254 nm。结果:毛蕊异黄酮葡萄糖苷、刺芒柄花苷、毛蕊异黄酮、山柰酚、异鼠李素和芒柄花素色谱峰均得到了良好地分离,分别在0.83~8.33、0.21~2.10、0.07~0.72、0.06~0.58、0.04~0.40和0.04~0.41μg·mL-1范围内线性良好,平均回收率结果为97.2%~101.6%,RSD为0.78%~2.4%。这6个黄酮类成分可能存在一定的规律。结论:方法简便、快速、准确,适用于黄芪中黄酮类成分的测定与分析。
Objective: To establish an UPLC method for simultaneous quantification of campanulin, onospin, calycosin, kaempferol, isorhamnetin and formononetin in Astragali Radix and analyze regular pattern of six flavonoids content data preliminarily. Methods: The analysis was performed on a Phenomenex Luna Omega C18(100 mm×2.1 mm, 1.6 μm) column at 35 ℃ using 0.1% formic acid solution and methanol as the mobile phase at a flow rate of 0.4 mL·min-1 with detection wavelength of 254 nm. Results: The separation of campanulin, onospin, calycosin, kaempferol, isorhamnetin and formononetin was good and the six types of flavonoids in 64 samples were quantified precisely. The calibration curves were linear in the ranges of 0.83-8.33 μg·mL-1, 0.21-2.10 μg·mL-1, 0.07-0.72 μg·mL-1, 0.06-0.58 μg·mL-1, 0.04-0.40 μg·mL-1 and 0.04-0.41 μg·mL-1 for campanulin, onospin, calycosin, kaempferol, isorhamnetin and formononetin, respectively. The mean recoveries of campanulin, onospin, calycosin, kaempferol, isorhamnetin and formononetin were 97.2%-101.6% with RSDs of 0.7%-2.4%. These six flavonoids might have some rules. Conclusion: This method is simple, rapid, accurate and is suitable for the analysis of flavonoids in Astragali Radix.
[1] 中华人民共和国药典2020年版.一部[S].2020:315
ChP 2020. Vol Ⅰ[S].2020:315
[2] DU HW, ZHAO XL, ZHANG AH, et al. Rapid separation, identification and analysis of Astragalus membranaceus Fisch. using liquid chromatography–tandem mass spectrometry[J].J Chromatogr Sci, 2014, 52(3):226
[3] LI K, GAO F, WANG G, et al. Identification of cultured and natural Astragalus root based on monosaccharide mapping[J].Molecules, 2015, 20(9):16466
[4] LI X, QU L, DONG Y, et al. A review of recent research progress on the Astragalus genus[J].Molecules, 2014, 19(11):18850
[5] LEE SM, JEONG JS, KWON HJ, et al. Quantification of isoflavonoids and triterpene saponins in Astragali Radix, the root of Astragalus membranaceus, via reverse-phase high-performance liquid chromatography coupled with integrated pulsed amperometric detection[J].J Chromatogr B, 2017, 1070:76
[6] 吴大正, 樊懿, 韩志芬, 等. 黄芪黄酮对低渗造成的血管内皮细胞单层通透性增高的改善作用[J].中药药理与临床, 2000, 16(4):16
WU DZ, FAN Y, HEN ZF, et al. Effect of flavonoids isolated from Astragalus membranaceus on the changes of endothelial permeability induced by low osmolarity[J].Pharmacol Clin Chin Mater Med, 2000, 16(4):16
[7] ZHANG LJ, LIU HK, HSIAO PC, et al, New isoflavonoid glycosides and related constituents from Astragali Radix(Astragalus membranaceus) and their inhibitory activity on nitric oxide production[J].J Agric Food Chem, 2011, 59(4):1131
[8] QI LW, YU QT, LI P, et al. Quality evaluation of Radix Astragali through a simultaneous determination of six major active isoflavonoids and four main saponins by high-performance liquid chromatography coupled with diode array and evaporative light scattering detectors [J].J Chromatogr A, 2006, 1134(1-2):162
[9] XIAO HB, KRUCKER M, ALBERT K, et al. Determination and identification of isoflavonoids in Radix Astragali by matrix solid-phase dispersion extraction and high-performance liquid chromatography with photodiode array and mass spectrometric detection[J].J Chromatogr A, 2004, 1032(1-2):117
[10] SONG JZ, HILLARY HW, QIAO CF, et al. Chemical comparison and classification of Radix Astragali by determination of isoflavonoids and astragalosides[J].J Pharm Biomed Anal, 2007, 47(2):406
[11] ZHENG YF, DUAN WP, SUN J, et al. Structural identification and conversion analysis of malonyl isoflavonoid glycosides in Astragali Radix by HPLC coupled with ESI-Q-TOF/MS[J].Molecules, 2019, 24(21):3929
[12] 张丽, 钱大玮, 卜凡淑, 等. 基于UPLC-MS的黄芪药材质量评价研究[J].药物分析杂志, 2020, 40(4):722
ZHANG L, QIAN DW, BU FS, et al. Study on quality evaluation of Astragali Radix based on UPLC-MS[J].Chin J Pharm Anal, 2020, 40(4):722
[13] 赵晨光, 李存玉, 杨珊, 等. 基于道地产区蒙古黄芪的质量差异性分析[J].中国中药杂志, 2020, 45(13):3183
ZHAO CG, LI CY, YANG S, et al. Analysis of quality difference based on Astragalus membranaceus var. mongholicus in genuine region[J].China J Chin Mater Med, 2020, 45(13):3183
[14] 裴文菡, 何凡, 程春松, 等. 中药黄芪质量评价方法的研究进展[J].中国现代应用药学, 2020, 37(5):620
PEI WH, HE F,CHENG CS, et al. Research progress on the quality evaluation methods of traditional Chinese medicine Astragali Radix[J].Chin J Mod Appl Pharm, 2020, 37(5):620
[15] 中华人民共和国药典2020年版. 四部[S].2020:482
ChP 2020. Vol Ⅳ[S].2020:482