Ultra high performance liquid chromatography coupled with triple quadrupole mass spectrometry and chemometric analysis of licorice based on the simultaneous determination of saponins and flavonoids

Licorice is among the most popular herbal medicines and frequently used in traditional medicine, food products, and cosmetics. In China, only Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat. and Glycyrrhiza glabra L. are officially used and are usually processed with honey prior to use. To maintain the quality of commercially available herbal products, a simple, rapid, and reliable ultra high performance liquid chromatography with triple quadrupole mass spectrometry was developed to investigate the major active constituents of commercially available licorice products. Nineteen components were accurately determined, including eight triterpenoid saponins, one triterpene, and ten flavonoids. Subsequently, multivariate statistical analysis methods were employed to further explore and interpret the experimental data. The results indicated that liquiritin apioside may be considered as a candidate index for the quality control of licorice as well as 18β‐glycyrrhizic acid and liquiritin. In addition, both 18β‐glycyrrhizic acid and licorice‐saponin G2 can be used for discrimination between crude and honey‐processed licorice. Furthermore, using 18β‐glycyrrhizic acid and liquiritin as markers, this work revealed that the quality of licorice products may have declined in recent years. This highlights the need for additional effort focused on good agricultural practice during the processing of licorice. In summary, this study provides a valuable reference for the quality assessment of licorice.     

Simultaneous separation of triterpenoid saponins and flavonoid glycosides from the roots of Glycyrrhiza uralensis Fisch by pH‐zone‐refining counter‐current chromatography

Glycosides including triterpenoid saponins and flavonoid glycosides are the main constituents of Glycyrrhiza uralensis Fisch (licorice) and exhibit prominent pharmacological activities. However, conventional methods for the separation of glycosides always cause irreversible adsorption and unavoidable loss of sample due to their high hydrophilicities. The present paper describes a convenient method for the simultaneous separation of triterpenoid saponins and flavonoid glycosides from licorice by pH‐zone‐refining counter‐current chromatography. Ethyl acetate/n‐butanol/water (2:3:5, v/v) with 10 mM TFA in the upper organic stationary phase and 10 mM ammonia in the lower aqueous mobile phase was used as the biphasic solvent system. Three triterpenoid saponins and two flavonoid glycosides including licorice‐saponin A3 (63.3 mg), glycyrrhizic acid (342.2 mg), 3‐O‐[β‐d ‐glucuronopyranosyl‐(1 → 2)‐β‐d ‐galactopyranosyl]glycyrrhetic acid (56.0 mg), liquiritin apioside (232.6 mg), and liquiritin (386.5 mg) were successfully obtained from licorice ethanol extract (2 g) in one step. This method subtly takes advantage of the common acidic properties of triterpenoid saponins and flavonoid glycosides, and obviously is much more efficient and convenient than the previous methods. It is also the first time that the separation of acidic triterpenoid saponins by using pH‐zone‐refining counter‐current chromatography has been reported.     

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.     

Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for the analysis of glycyrrhizic acid

Glycyrrhizic acid (GL) is a major active compound of licorice. The specific monoclonal antibody (MAb) (designated as 8F₈A₈H₄2H₇) against GL was produced with the immunogen GL–BSA conjugate. The dissociation constant (K _d) value of the MAb was approximately 9.96×10⁻¹⁰ M. The cross reactivity of the MAb with glycyrrhetic acid was approximately 2.6%. The conventional indirect competitive enzyme-linked immunosorbent assay (icELISA) and simplified icELISA adapted with a modified procedure were established using the MAb. The IC₅₀ value and the detect range by the conventional icELISA were 1.1 ng mL⁻¹ and 0.2–5.1 ng mL⁻¹, respectively. The IC₅₀ value and the detect range by the simplified icELISA were 5.3 ng mL⁻¹ and 1.2–23.8 ng mL⁻¹, respectively. The two icELISA formats were used to analyze GL contents in the roots of wild licorice and different parts of cultivated licorice (Glycyrrhiza uralensis Fisch). The results obtained with the two icELISAs agreed well with those of the HPLC analysis. The correlation coefficient was more than 0.98 between HPLC and the two icELISAs. The two icELISAs were shown to be appropriate, simple, and effective for the quality control of raw licorice root materials.     

Metabolic routes along digestive system of licorice: multicomponent sequential metabolism method in rat

This study was conducted to establish the multicomponent sequential metabolism (MSM) method based on comparative analysis along the digestive system following oral administration of licorice (Glycyrrhiza uralensis Fisch., leguminosae), a traditional Chinese medicine widely used for harmonizing other ingredients in a formulae. The licorice water extract (LWE) dissolved in Krebs–Ringer buffer solution (1 g/mL) was used to carry out the experiments and the comparative analysis was performed using HPLC and LC‐MS/MS methods. In vitro incubation, in situ closed‐loop and in vivo blood sampling were used to measure the LWE metabolic profile along the digestive system. The incubation experiment showed that the LWE was basically stable in digestive juice. A comparative analysis presented the metabolic profile of each prototype and its corresponding metabolites then. Liver was the major metabolic organ for LWE, and the metabolism by the intestinal flora and gut wall was also an important part of the process. The MSM method was practical and could be a potential method to describe the metabolic routes of multiple components before absorption into the systemic blood stream. Copyright © 2015 John Wiley & Sons, Ltd.     

Flavonoids of Roots of Glycyrrhiza uralensis Growing in Siberia

The composition of phenolic components of roots and rhizomes ofGlycyrrhiza uralensisFisch. from Siberia is studied. A total of 15 components belonging to 8 structural types including 4' -methylglucoliquiritigenin, which was previously unknown in licorices, is found. The components in samples collected from various regions of Siberia are practically identical.     

Isolation and GC-MS determination of flavonoids from Glycyrrhiza glabra root

Components of the ethylacetate extract of Glycyrrhiza glabra root were separated based on their acid-base properties. The major components of the fraction soluble in aqueous NaOH were identified by GC-MS as glabridin, the principal component of the ethylacetate extract of licorice root (Glycyrrhiza glabra L.), 4-O-methylglabridin, and hispaglabridin B. The isoflavene glabrin was identified in addition to the isoflavan derivatives. __________ Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 236–240, May–June, 2006.     

Preparative isolation and analysis of alcohol dehydrogenase inhibitors from Glycyrrhiza uralensis root using ultrafiltration combined with high‐performance liquid chromatography and high‐speed countercurrent chromatography

A simple, rapid, and effective assay based on ultrafiltration combined with high‐performance liquid chromatography and high‐speed countercurrent chromatography was developed for screening and purifying alcohol dehydrogenase inhibitors from Glycyrrhiza uralensis root extract. Experiments were carried out to optimize binding conditions including alcohol dehydrogenase concentration, incubation time, temperature, and pH. By comparing the chromatograms, three compounds were found possessing alcohol dehydrogenase binding activity in Glycyrrhiza uralensis root. Under the target‐guidance of ultrafiltration combined with the high‐performance liquid chromatography experiment, liquiritin ( 1 ), isoliquiritin ( 2 ), and liquiritigenin ( 3 ) were separated by high‐speed countercurrent chromatography using ethyl acetate/methanol/water (5:1:4) as the solvent system. The alcohol dehydrogenase inhibitory activities of these three isolated compounds were assessed; compound 2 showed strongest inhibitory activity with an IC₅₀ of 8.95 μM. The results of the present study indicated that the combinative method using ultrafiltration, high‐performance liquid chromatography and high‐speed countercurrent chromatography could be widely applied for the rapid screening and isolation of enzyme inhibitors from complex mixtures.     

Chemical interaction between Paeonia lactiflora and Glycyrrhiza uralensis,the components of Jakyakgamcho‐tang,using a validated high‐performance liquid chromatography method: Herbal combination and chemical interaction in a decoction

The herbal combination is the basic unit of a herbal formula that affects the chemical characteristics of individual herbs. In the present study, a method of simultaneous determination of the 11 marker compounds in Jakyakgamcho‐tang was developed using high‐performance liquid chromatography with photodiode array detection. The validated analytical method was successfully applied to approach the chemical interaction between Paeonia lactiflora and Glycyrrhiza uralensis in co‐decoction. In P. lactiflora, the contents of gallic acid, oxypaeoniflorin, (+)‐catechin, paeoniflorin, and benzoylpaeoniflorin were decreased, while those of albiflorin and benzoic acid were increased; in G. uralensis, the contents of liquiritin, isoliquiritin, ononin, and glycyrrhizin were decreased, when decocting two herbs together. Moreover, as the ratio between P. lactiflora and G. uralensis was increased, the contents of chemical contents from each herb were proportionally increased. However, each content of marker compound per the gram of herbal medicine was decreased as the ratio of combinative herbs increased. The results showed that P. lactiflora and G. uralensis affect the extraction efficiency of chemical compounds in a Jakyakgamcho‐tang decoction. Overall, the method established in this study was simple, rapid, and accurate, and would be useful for the determination of marker compounds and for the investigation of the chemical interaction between herbal medicines.     

Quantitative analysis of multicomponents by a single marker and quality evaluation of Venenum Bufonis from different geographical origins

Bufadienolides are the main bioactive components of Venenum Bufonis (VB) and have been widely used to treat different types of human cancers for decades. The bufadienolide content in VB varies significantly in materials from different geographical origins. In this work, a new strategy for the quality assessment of VB was developed through quantitative analysis of multi‐components by single marker (QAMS). Cinobufagin was selected as the internal reference substance; seven bufadienolides were separated and simultaneously determined based on relative correction factors. The correlation coefficient value (r ≥ 0.9936) between QAMS and the normal external standard method proved the consistency of the two methods. According to the outcomes of 30 batches of VB samples, the contents of the seven bufadienolides were used for further chemometric analysis. All of the samples of VB from various geographical origins were divided into three categories based on hierarchical cluster analysis and radar plot, which indicated the crucial influence of geographical origins on VB. This study showed that QAMS combined with chemometristry could be used to comprehensively evaluate and effectively control the quality of VB from different geographical origins.     

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.     

Accurate discrimination of Gastrodia elata from different geographical origins using high‐performance liquid chromatography fingerprint combined with boosting partial least‐squares discriminant analysis

Gastrodia elata from different geographical origins varies in quality and pharmacological activity. This study focused on the classification and identification of Gastrodia elata from six producing areas using high‐performance liquid chromatography fingerprint combined with boosting partial least‐squares discriminant analysis. Before recognition analysis, a principal component analysis was applied to ascertain the discrimination possibility with high‐performance liquid chromatography fingerprints. And then, boosting partial least‐squares discriminant analysis and conventional partial least‐squares discriminant analysis were applied in this study. Experimental results indicated that the adaptive iteratively reweighted penalized least‐squares algorithm could eliminate the baseline drift of high‐performance liquid chromatography chromatograms effectively. And compared with partial least‐squares discriminant analysis, the total recognition rates using high‐performance liquid chromatography fingerprint combined with boosting partial least‐squares discriminant analysis for the calibration sets and prediction sets were improved from 94 to 100% and 86 to 97%, respectively. In conclusion, high‐performance liquid chromatography combined with boosting partial least‐squares discriminant analysis, which has such advantages as effective, specific, accurate, non‐polluting, has an edge for discrimination of traditional Chinese medicine from different geographical origins. And the proposed methodology is a useful tool to classify and identify Gastrodia elata from different geographical origins.     

Establishment and Quality Assessment of Tissue Cultures in Glycyrrhiza uralensis Fisch.

In this study, seedling, callus, cell, and adventitious root of Glycyrrhiza uralensis Fisch. have been established. In order to find the best one for producing G. uralensis active constituents, triterpenoid saponins and flavonoids in native root and tissue cultures were determined, and the contents in different G. uralensis materials were analyzed using cluster analysis. The contents of triterpenoid saponins and glycyrrhizic acid in tissue cultures were much lower than that in native G. uralensis. The total flavonoids content we determined in adventitious root was 6.31 mg?g^?1, which was close to that of native root (9.82 mg?g^?1). Based on the cluster analysis, we found that G. uralensis cultures were not suitable for production of glycyrrhizic acid, while adventitious root had a greater capability of flavonoids production comparing to seedling, callus, and cell.     

GC–MS combined with chemometric techniques for the quality control and original discrimination of Curcumae longae rhizome: Analysis of essential oils

Curcumae longae rhizome is a widely used traditional herb in many countries. Various geographical origins of this herb might lead to diversity or instability of the herbal quality. The objective of this work was to establish the chemical fingerprints for quality control and find the chemical markers for discriminating these herbs from different origins. First, chemical fingerprints of essential oil of 24 C. longae rhizome from four different geographical origins in China were determined by GC–MS. Then, pattern recognition techniques were introduced to analyze these abundant chemical data in depth; hierarchical cluster analysis was used to sort samples into groups by measuring their similarities, and principal component analysis and partial least‐squares discriminate analysis were applied to find the main chemical markers for discriminating these samples. Curcumae longae rhizome from Guangxi province had the highest essential oil yield (4.32 ± 1.45%). A total of 46 volatile compounds were identified in total. Consistent results were obtained to show that C. longae rhizome samples could be successfully grouped according to their origins, and turmerone, ar‐turmerone, and zingiberene were the characteristic components for discriminating these samples of various geographical origins and for quality control. This finding revealed that fingerprinting analysis based on GC–MS coupled with chemometric techniques could provide a reliable platform to discriminate herbs from different origins, which is a benefit for quality control.     

Geographical Authentication of Gentiana Rigescens by High-Performance Liquid Chromatography and Infrared Spectroscopy

The chemical characteristics of Gentiana rigescens are extremely variable due to their geographical origins which should be determined to evaluate the quality of this species. Different with other herbs with official tissue for classification materials, the geographical characterization of raw herbal materials on the basis of nonmedicinal parts is rarely discussed. Chromatographic active components were used as references to characterize the chemical profiles of samples from various geographical origins. Based on spectra data matrix of different botanical parts, the chemometric methods of partial least square discrimination analysis and support vector machine discrimination analysis were used to develop mathematical models to classify samples from different geographical origins. In terms of six active components, we found that significant differences were present in the tissue of G. rigescens based on geographical origins. In addition, the region with higher content of gentiopicroside was selected to be the optimal cultivated location. Chemometric results indicated that leaves were the optimal material for geographical characterization of G. rigescens with 100% accuracy by support vector machine while the accuracies of roots, stems, and flowers were 90.91, 96.10, and 97.01%, respectively. Partial least square discrimination analysis showed that accuracy values for roots, stems, leaves, and flowers were 35.65, 67.53, 76.62, and 50.75%, respectively, which also indicated that leaves are the optimal material. In conclusion, northwest Yunnan Province with higher content of gentiopicroside was selected to be the optimal cultivation location. Furthermore, leaves should be used for the most accurate geographical authentication.     

Study of two-dimensional liquid chromatography with high temperature NPLC and room temperature RPLC

To overcome the peak band broadening and to increase the peak capacity and separation efficiency of a two-dimensional liquid chromatographic system, a high-temperature normal phase liquid chromatography (HTNPLC) was used as the first dimension (1^st-D), and a RPLC was used as the second dimension (2^nd-D). The sample was first separated on the 1^st-D CN column and the primary eluent stored in the sampling-loop system alternatively (in HTNPLC×RPLC mode) or selectively (in HTNPLC/RPLC mode) and was then transferred to 2^nd-D C₁₈ column for further separation. The resolution and separation efficiency of the systems were greatly improved. The systems were evaluated by analyzing several polycyclic aromatic hydrocarbons and Glycyrrhiza uralensis extract. __________ Translated from Journal of Instrumental Analysis, 2008, 27(1) (in Chinese)     

Quantification and bio-assay of α-glucosidase inhibitors from the roots of Glycyrrhiza uralensis Fisch.

This work aimed to investigate the α-glucosidase inhibitor from the roots of Glycyrrhiza uralensis Fisch. Seven flavonoids were isolated, and the total content of compounds 1–7 were more than 50% in Glycyrrhiza total flavones (GTF), and the content of compound 1 and 2 was abundant in GTF. The results of the α-glucosidase inhibitory activities indicated that compounds 1–6 and GTF respectively with the IC₅₀ values of 31.303, 30.263, 23.363, 19.528, 10.854, 26.454 and 21.641 μg/mL exhibited the more potent activity than acarbose with the IC₅₀ values of 38.995 μg/mL. These result suggested that Glycyrrhiza flavonoids may become a valid alternative of potential basis for new hypoglycaemic and antidiabetic agents.     

Structural characterization and identification of oleanane-type triterpene saponins in Glycyrrhiza uralensis Fischer by rapid-resolution liquid chromatography coupled with time-of-flight mass spectrometry

Oleanane‐type triterpene saponins (OTS) are major active ingredients in Glycyrrhiza uralensis. In this work, a rapid‐resolution liquid chromatography with time‐of‐flight mass spectrometry (RRLC/TOF‐MS) method has been developed to characterize and identify OTS from G. uralensis. The major diagnostic ions and fragmentation pathways from thirteen OTS have been characterized for the first time. At a low fragmentor voltage of 120 V in positive ion mode, the precursor ion [M + H]⁺ or/and [M + Na]⁺ was obtained for accurate determination of molecular formula. When the fragmentor voltage was increased to 425 V, abundant characteristic fragment ions were observed for structural characterization. Neutral losses of sugar moieties, such as glucuronic acid (GlcA, 176 Da), glucose (Glc, 162 Da) and rhamnose (Rha, 146 Da), were commonly observed in the MS spectra for prediction of the sugar number and sequences. Other typical losses included AcOH (60 Da), CH₂O (30 Da), 2 × H₂O (2 × 18 Da) and HCOOH (46 Da) from [Aglycone + H–H₂O]⁺ (named [B]⁺), corresponding to the presence of a C₂₂‐acetyl group, C₂₄‐hydroxyl group, C₂₂‐hydroxyl group or C₃₀‐carboxyl group on the aglycone moiety, respectively. In particular, characteristic ring cleavages of the aglycone moieties on A‐ and B‐rings were observed. Based on the fragmentation patterns of reference compounds, nineteen OTS have been identified in an extract of G. uralensis, thirteen of which were unambiguously identified and the other six were tentatively assigned. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of trace elements in Chinese medicinal plants by instrumental neutron activation analysis

Roots of Astragalus membranaceus, Angelica sinensis, Glycyrrhiza uralensis and Codonopsis pilosula, which were often used as herbs in traditional Chinese medicine, were analyzed by Instrumental Neutron Activation Analysis (INAA). The samples were collected in Gansu, northwest of China and irradiated at the 15 MW heavy water reactor in China Institute of Atomic Energy (CIAE). The induced activities were counted by a well calibrated low background γ-spectrometer equipped with a high efficiency coaxial high-purity germanium (HPGe) detector. The concentrations of eighteen trace elements (Ca, Fe, Na, Zn, Ba, Rb, Ce, Cr, La, Co, Th, Cs, Sb, Sc, Sm, Hf, Eu and Tb) in the herbs were determined. Possible links between pharmacological action of the herbs and content of some elements were also discussed in this paper. The measured results were compared with the reported values in literature.     

Deciphering the chemical composition of Ganoderma lucidum from different geographical origins by mass spectrometry molecular networking coupled with multivariate analysis

Ganoderma lucidum is a medicinal fungus that has been widely used in China and many Asian countries for thousands of years. This once rare macrofungus has now been artificially cultivated in a number of regions in China. However, detailed knowledge of its composition across different geographical origins is still lacking, as are analytical methods for comprehensive profiling of the diverse phytochemicals contained in G. lucidum. In this work, an on-demand strategy based on high-resolution MS and molecular networking is applied for natural product characterization, which led to the identification of 84 constituents in G. lucidum. Moreover, multivariate analysis, including hierarchical cluster analysis and orthogonal partial least squares-discriminant analysis, was used to analyze the (dis)similarity of the G. lucidum samples collected from the three main production areas (i.e., Jilin, Henan and Shandong Province). The results revealed a significant variation in the chemical composition of samples from different provinces. Marker constituents corresponding to the differentiation were then screened in terms of the variable importance in projection value, P-value and fold change. A total of 24 constituents were identified as geoherbalism markers, such as ganoderenic acid A for Henan, ganolucidic acid B for Jilin and ganodernoid D for Shandong. This proof-of-concept application demonstrates that combining MS molecular networking with meticulous multivariate analysis can provide a sensitive and comprehensive analytical approach for the quality assessment of traditional Chinese medicine ingredients. This study also suggests that the bioactivity and efficacy from different origins should be further evaluated considering the large difference in chemical compositions.