Metabolite profiling of licorice (Glycyrrhiza glabra) from different locations using comprehensive two-dimensional liquid chromatography coupled to diode array and tandem mass spectrometry detection

Profiling of the main metabolites from several licorice (Glycyrrhiza glabra) samples collected at different locations is carried out in this work by using comprehensive two-dimensional liquid chromatography (LC × LC) coupled to diode array (DAD) and mass spectrometry (MS) detectors. The optimized method was based on the application of a HILIC-based separation in the first dimension combined with fast RP-based second dimension separation. This set-up was shown to possess powerful separation capabilities allowing separating as much as 89 different metabolites in a single sample. Identification and grouping of metabolites according to their chemical class were achieved using the DAD, MS and MS/MS data. Triterpene saponins were the most abundant metabolites followed by glycosylated flavanones and chalcones, whereas glycyrrhizic acid, as expected, was confirmed as the main component in all the studied samples. LC × LC-DAD-MS/MS was able to resolve these complex licorice samples providing with specific metabolite profiles to the different licorice samples depending on their geographical origin. Namely, from 19 to 50 specific compounds were exclusively determined in the 2D-chromatograms from the different licorice samples depending on their geographical origin, which can be used as a typical pattern that could potentially be related to their geographical location and authentication.     

微波辅助提取甘草中甘草苷的工艺研究

以乙醇溶液为溶剂微波辅助提取甘草中甘草苷,紫外可见分光光度法测定甘草苷的含量。以甘草苷的提取率为评价指标,考察微波辐射时间、料液比、乙醇浓度、微波功率等因素,利用正交设计实验,筛选最佳工艺条件。结果表明最佳工艺条件为：微波辐射时间40min、料液比为1∶12（g∶mL）、乙醇浓度为40%、微波功率640W。与传统回流提取方法相比,微波辅助提取快速,甘草苷的提取率较高。     

Rapid determination of flavonoids in licorice and comparison of three licorice species

A simple, sensitive and fast method for the simultaneous quantitation of 15 flavonoids in licorice based on an ultra high performance liquid chromatography coupled with triple quadrupole electrospray tandem mass spectrometry had been established and validated in this study. The analysis was performed on an ACQUITY HSS T3 column with gradient elution using a mobile phase consisted of A (0.1% formic acid in water)/B (acetonitrile) at a flow rate of 0.4 mL/min. Satisfactory separation of these compounds was obtained in less than 9 min. All calibration curves showed good linearity (r² = 0.9940) during the test ranges. The precision, repeatability, accuracy, limit of detection and limit of quantification were also fully investigated. The validated method was successfully applied for the simultaneous quantitation of 15 flavonoids in 106 licorice samples which contained 83 batches of G. uralensis, 14 batches of G. glabra and 9 batches of G. inflata. Furthermore, multivariate statistical analysis (using principal components analysis) was performed to classify the samples based on the contents of the 15 analyzed compounds. The results showed that all of these licorice samples were rich in flavonoids, although their contents were obviously various, and the proposed method could serve as a prerequisite for quality control of licorice products.     

A green and efficient protocol for large‐scale production of glycyrrhizic acid from licorice roots by combination of polyamide and macroporous resin adsorbent chromatography

A green and efficient method for large‐scale preparation of glycyrrhizic acid from licorice roots was developed by combination of polyamide and macroporous resin. The entire preparation procedure consisted of two simple separation steps. The first step is to use polyamide resin to remove licorice flavoniods from the licorice crude extract. Subsequently, various macroporous resins were tried to purify glycyrrhizic acid, and HPD‐400 showed the most suitable adsorption and desorption properties. Under the optimized conditions, a large‐scale preparation of glycyrrhizic acid from licorice roots was carried out. A 20 kg raw material produced 0.43 kg of glycyrrhizic acid using green aqueous ethanol as the solvent. The purity of glycyrrhizic acid was increased from 11.40 to 88.95% with a recovery of 76.53%. The proposed method may be also extended to produce large‐scale other triterpenoid saponins from herbal materials.     

Screening and characterization of natural antioxidants in four Glycyrrhiza species by liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry

Licorice, derived from the dried roots and rhizomes of several species of genus Glycyrrhiza L. (Leguminosae family), has been traditionally used in herbal medicine for over 4000 years. In recent years, the interest in antioxidative constituents in licorice has greatly increased. In this work, a new method based on 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) spiking test combined with HPLC coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) analysis was developed to screen and identify the antioxidants in licorice. The results of the method validation indicated that the developed method was reliable and repeatable. Compared with DPPH on-line method, the HPLC-Q-TOF MS/MS method combined with DPPH spiking test offered much higher sensitivity and resolution. Using this method, 35 radical scavengers were screened from four Glycyrrhiza species (G. inflata, G. glabra, G. pallidiflora and G. uralensis), and 21 of them were unambiguously or tentatively identified by HPLC-Q-TOF MS/MS. Among the 21 identified flavonoids, 10 compounds had been reported to possess antioxidative activities in the previous studies, and the radical scavenging activities of the other 11 compounds were reported for the first time. The effects of six purified flavonoids on DPPH radical and lipid peroxidation were evaluated for validation of the developed method. The results indicated that HPLC-Q-TOF MS/MS coupled with DPPH treatment is an efficient and powerful method to discover the potential antioxidative compounds from the complex natural product mixtures. In this study, the identified components with free radical scavenging activity, would help to explain the therapeutic benefit of licorice in the treatment of human disease associated with oxidative stress.     

Purification of flavonoids and triterpene saponins from the licorice extract using preparative HPLC under RP and HILIC mode

A four‐channel preparative HPLC was employed to isolate and purify compounds from licorice extract. Two separation modes, RP and hydrophilic interaction LC (HILIC), were used in preparative HPLC. HILIC mode was adopted to resolve the purification of the compounds with similar hydrophobicity, which were co‐eluted under RP mode. Using the two separation modes during the purification process, fifteen compounds were isolated from licorice extract. The results indicated that preparative HPLC performed under HILIC mode is an efficient method for the isolation and purification of compounds from natural products.     

Chemical analysis of the Chinese herbal medicine Gan-Cao (licorice)

Gan-Cao, or licorice, is a popular Chinese herbal medicine derived from the dried roots and rhizomes of Glycyrrhiza uralensis, G. glabra, and G. inflata. The main bioactive constituents of licorice are triterpene saponins and various types of flavonoids. The contents of these compounds may vary in different licorice batches and thus affect the therapeutic effects. In order to ensure its efficacy and safety, sensitive and accurate methods for the qualitative and quantitative analyses of saponins and flavonoids are of significance for the comprehensive quality control of licorice. This review describes the progress in chemical analysis of licorice and its preparations since 2000. Newly established methods are summarized, including spectroscopy, thin-layer chromatography, gas chromatography, high-performance liquid chromatography (HPLC), liquid chromatography/mass spectrometry (LC/MS), capillary electrophoresis, high-speed counter-current chromatography (HSCCC), electrochemistry, and immunoassay. The sensitivity, selectivity and powerful separation capability of HPLC and CE allows the simultaneous detection of multiple compounds in licorice. LC/MS provides characteristic fragmentations for the rapid structural identification of licorice saponins and flavonoids. The combination of HPLC and LC/MS is currently the most powerful technique for the quality control of licorice.     

Application of analytical chemistry in the quality evaluation of Glycyrrhiza Spp

Licorice, one of the oldest traditional Chinese medications, is widely used in the treatment of various diseases. With the development of science and technology, an increasing amount of analytical techniques have been applied to the quality control of licorice. Herein, we summarize several of the quality control methods developed in recent years. These approaches include sample preparation processes, high-performance thin layer chromatography, gas chromatography, gas chromatography/mass spectrometry, high-performance liquid chromatography, and liquid chromatography/mass spectrometry (LC/MS). Among these various techniques, LC/MS has come forward as one of the main methods forquality control of licorice.     

乌拉尔甘草单粒种子硬实特性的近红外光谱分析

以乌拉尔甘草种子为材料,采用近红外光谱结合定性偏最小二乘法对244粒种子(硬实种子和非硬实种子比例为1∶1)的硬实性进行了鉴别研究,并特制一样品杯用于单粒种子的光谱采集,以降低人为误差。研究结果表明,4次重复平均光谱所建模型鉴别率显著高于单次光谱所建模型,光谱范围采用4 000～8 000 cm-1时模型效果较好,校正集、检验集、预测集样本的鉴别率分别为95.53%,95.94%和94.53%,采用不同建模样品所建模型其预测准确率均在90%以上,硬实种子和非硬实种子的预测准确率分别为92.50%和96.56%。种子大小和颜色均会影响模型的鉴别率,种子颜色的影响相对更大。     

Simultaneous determination of five flavonoids in licorice using pressurized liquid extraction and capillary electrochromatography coupled with peak suppression diode array detection

Pressurized liquid extraction (PLE) and capillary electrochromatography (CEC) methods were developed for the simultaneous determination of five flavonoids, namely liquiritin, isoliquiritin, ononin, liquiritigenin and isoliquiritigenin, in licorice using baicalein as internal standard (IS). Peak suppression technique was used for the quantification of ononin because of its poor resolution with isoliquiritin. The analysis was performed on a Hypersil C₁₈ capillary (3 μm, 100 μm/25 cm) with a mixture of 10 mM phosphate buffer (pH 3.0)/ACN (65:35, v/v) as mobile phase running at 25 kV and 30 °C. The detection wavelengths were set at 275 nm (without reference wavelength for liquiritin and liquiritigenin), 360 nm (without reference wavelength for isoliquiritin and isoliquiritigenin) and 254 nm (with reference wavelength of 405 nm for ononin). All calibration curves showed good linearity (R² > 0.9993) within the test ranges. The LOD and LOQ were lower than 2.1 and 8.3 μg/mL, respectively. The RSDs of intra- and interday for relative peak areas of five analytes to IS were less than 3.8 and 4.7%, respectively, and the recoveries were 98.2–103.8%. The validated method was successfully applied to the quantitative analysis of five flavonoids in licorice, which is helpful to its quality control.