3种不同基原甘草中4个主要黄酮类化合物的含量分析

Simultaneous determination of liquiritin,isoliquiritin,liquiritigenin and isoliquiritigenin in Glycyrrhiza uralensis Fisch.,Glycyrrhiza glabra L.,and Glycyrrhiza inflata Bat. by HPLC

目的:对中国药典规定的3种不同基原甘草样品中甘草苷、异甘草苷、甘草素和异甘草素的含量进行分析比较。方法:以栽培于同一产地的3种不同基原两年生甘草作为实验材料,HPLC法对材料中的甘草苷、异甘草苷、甘草素和异甘草素含量进行测定。采用CAPCELL PAK C_(18) MGII(4.6 mm×250 mm,5μm)色谱柱,以乙腈(A)-0.1%磷酸溶液(B)为流动相,梯度洗脱,流速为1.0 m L·min^(-1),检测波长为276 nm(0~13 min,检测甘草苷)、360 nm(13~23 min,检测异甘草苷)、276 nm(23~28 min,检测甘草素)、376 nm(28~55 min,检测异甘草素),柱温为30℃。结果:甘草苷、异甘草苷、甘草素和异甘草素分离良好,线性范围分别为1.15×10^(-2)~0.230μg(r=1.000)、4.45×10^(-3^)8.90×10^(-2)μg(r=1.000)、1.15×10^(-3^)2.30×10^(-2)μg(r=0.998)、2.27×10^(-3^)4.54×10^(-2)μg(r=1.000),检测限和定量限依次为1.13 ng和3.42 ng、0.896 ng和2.70 ng、0.463 ng和1.39 ng、0.454 ng和1.38 ng。本方法灵敏度、精密度、准确性、重复性、回收率、耐用性均良好。在3种基原的甘草样品中,4个黄酮类成分的含量存在极显著差异(P<0.01),甘草苷、异甘草苷、甘草素、异甘草素在乌拉尔甘草中的含量均最高,分别为(^(-2)0.75±0.524)mg·g1、(4.453±0.057)mg·g^(-1)、(0.610±0.019)mg·g^(-1)和(0.272±0.008)mg·g^(-1),光果甘草次之,含量分别为(6.623±0.405)mg·g^(-1)、(1.562±0.053)mg·g^(-1)、(0.325±0.036)mg·g^(-1)和(0.180±0.012)mg·g^(-1),胀果甘草最低,含量分别为(2.700±0.232)mg·g^(-1)、(0.821±0.042)mg·g^(-1)、(0.153±0.006)mg·g^(-1)和(0.115±0.005)mg·g^(-1);甘草苷与异甘草苷、甘草素与异甘草素的含量在3种不同基原甘草样品中均存在极显著相关关系(P<0.01)。结论:本文建立的HPLC方法经方法学验证,可用于甘草中甘草苷、异甘草苷、甘草素和异甘草素的含量分析,并为甘草的质量控制及以黄酮类化合物为目标的优质甘草筛选与定向育种提供科学依据。

Objective：To compare the contents of liquiritin,isoliquiritin,liquiritigenin and isoliquiritigenin in the three original plants of licorice stipulated in Chinese pharmacopoeia. Methods：Three original plants of licorice cultured in the same condition for two years were collected as study samples. The contents of liquiritin,isoliquiritin,liquiritigenin and isoliquiritigenin were analyzed by an HPLC method using chromatographic column CAPCELL PAK C18 MGII（250 mm×4.6 mm,5 μm） at the column temperature of 30 ℃. The mobile phase consisted of acetonitrile（A） and 0.1% phosphoric acid（B） with gradient elution at a flow rate of 1.0 m L·min-1. The UV detection wavelength was 276 nm（0-13 min） for liquiritin,360 nm（13-23 min） for isoliquiritin,276 nm（23-28 min） for liquiritigenin,and 376 nm（28-55 min） for isoliquiritigenin,respectively. Results：The calibration curves for liquiritin,isoliquiritin,liquiritigenin and isoliquiritigenin were Y=2×10^6X-2×10^（-1）（r=1.000）,Y=4×10^6X＋3×10^（-1）（r=1.000）,Y=3×10^6X-1×10^（-1）（r=0.998）,and Y=5×10^6X＋2×10^（-1）（r=1.000）,respectively,which reflected a good linearity in the range of 1.15×10^（-2）-0.230 μg,4.45×10^（-3）-8.90×10^（-2） μg,1.15×10^（-3）-2.30×10^（-2） μg and 2.27×10^（-3）-4.54×10^（-2） μg. The LOD and LOQ were 1.13 ng and 3.42 ng,0.896 ng and 2.70 ng,0.463 ng and 1.39 ng,and 0.454 ng and 1.38 ng for liquiritin,isoliquirtin,liquiritigenin and isoliquiritigenin respectively.The sensitivity,accuracy,precision,repeatability,durability and the average recoveries all met the requirements. It was demonstrated that the contents of liquiritin,isoliquiritin,liquiritigenin and isoliquiritigenin in three origins of licorice varied a lot（P 〈0.01）. The highest contents of 4 compounds were found in Glycyrrhiza uralensis Fisch.,determined as（20.75±0.524） mg·g-1,（4.453±0.057）mg·g-1,（0.610±0.019） mg·g-1 and（0.272±0.008） mg·g-1,respectively,and intermediate in Glycyrrhiza glabra L.,shown as（6.623±0.405） mg·g-1,（1.562±0.053） mg·g-1,（0.325±0.036） mg·g-1 and（0.180±0.012）mg·g-1 respectively,and lowest in Glycyrrhiza inflata Bat.,which were（2.700±0.232） mg·g-1,（0.821±0.042） mg·g-1,（0.153±0.006） mg·g-1 and（0.115±0.005） mg·g-1,respectively. Furthermore,the content of liquiritin was highly correlated to the content of isoliquiritin,and the content of liquiritigenin was highly correlated to the content of isoliquiritigenin in all three origins of licorice（P 〈0.01）. Conclusion：The developed method is proved suitable for liquiritin,isoliquiritin,liquiritigenin and isoliquiritigenin determination in the three original plants of licorice,and for the quality control of licorice,providing instructions in directed breeding aiming at flavonoids.