Min Ye

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.

Phytochemical analysis of traditional Chinese medicine using liquid chromatography coupled with mass spectrometry

Traditional Chinese medicine (TCM) is commonly considered to operate due to the synergistic effects of all the major and minor components in the medicines. Hence sensitive and comprehensive analytical techniques are needed to acquire a better understanding of the pharmacological basis of the herb and to enhance the product quality control. The present review mainly focuses on the phytochemical analysis of TCMs using high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS). Atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) are the two commonly used ion sources. Triple quadrupole, ion trap (IT), Fourier transform ion cyclotron resonance (FTICR) and time-of-flight (TOF) mass spectrometers are used as on-line analyzer. The relationship between structural features and fragmentation patterns should be investigated as thoroughly as possible and hence be applied in the on-line analysis to deduce the structures of detected peaks. Characteristic fragmentation behaviors of the reference standards, as well as information regarding polarity obtained from retention time data, on-line UV spectra, data from the literature and bio-sources of the compounds allowed the identification of the phytochemical constituents in the crude extracts. Although a mass spectrometer is not a universal detector, high-performance liquid chromatography coupled with multistage mass spectrometry (HPLC-MS(n)) technique was still proved to be a rapid and sensitive method to analyze the majority of the many constituents in herbal medicines, particularly for the detection of those present in minor or trace amounts. The methods established using HPLC-MS techniques facilitate the convenient and rapid quality control of traditional medicines and their pharmaceutical preparations. However, the quantitative analysis is not the topic of this review.

A practical strategy for the characterization of coumarins in Radix Glehniae by liquid chromatography coupled with triple quadrupole-linear ion trap mass spectrometry.

The aim of the present study was to develop a practical method for the characterization of coumarins in Radix Glehniae by liquid chromatography-mass spectrometry (LC-MS). First, 10 coumarin standards (including two pairs of isomers) were studied, and mass spectrometry fragmentation patterns and elution time rules for the coumarins were found. Then, an extract of Radix Glehniae was analyzed by the combination of two scan modes, i.e., multiple ion monitoring-information-dependent acquisition-enhanced product ion mode (MIM-IDA-EPI) and precursor scan information-dependent acquisition-enhanced product ion mode (PREC-IDA-EPI) on a hybrid triple quadrupole-linear ion trap mass spectrometer. A total of 41 coumarins were identified on the basis of their mass spectrometry fragmentation patterns. This is the first time that these two scan modes have been combined to characterize chemical constituents in traditional Chinese medicine. This new method allowed the identification of coumarins in Radix Glehniae in trace amounts. The methodology proposed in this study could be valuable for the structural characterization of coumarins from complex natural and synthetic sources.

A sesquiterpene lactone antrocin from Antrodia camphorata negatively modulates JAK2/STAT3 signaling via microRNA let-7c and induces apoptosis in lung cancer cells.

Lung cancer is the leading cause of cancer deaths worldwide and current therapies fail to treat this disease in majority of cases. Antrodia camphorata is a medicinal mushroom being widely used as food dietary supplement for cancer prevention. The sesquiterpene lactone antrocin is the most potent among >100 secondary metabolites isolated from A. camphorata. However, the molecular mechanisms of antrocin-mediated anticancer effects remain unclear. In this study, we found that antrocin inhibited cell proliferation in two non-small-cell lung cancer cells, namely H441 (wild-type epidermal growth factor receptor, IC50 = 0.75 μM) and H1975 (gefitnib-resistant mutant T790M, IC50 = 0.83 μM). Antrocin dose dependently suppressed colony formation and induced apoptosis as evidenced by activated caspase-3 and increased Bax/Bcl2 ratio. Gene profiling studies indicated that antrocin downregulated Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway. We further demonstrated that antrocin suppressed both constitutively activated and interleukin 6-induced STAT3 phosphorylation and its subsequent nuclear translocation. Such inhibition is found to be achieved through the suppression of JAK2 and interaction between STAT3 and extracellular signal-regulated kinase. Additionally, antrocin increased microRNA let-7c expression and suppressed STAT signaling. The combination of antrocin and JAK2/STAT3 gene silencing significantly increased apoptosis in H441 cells. Such dual interruption of JAK2 and STAT3 pathways also induced downregulation of antiapoptotic protein mcl-1 and increased caspase-3 expression. In vivo intraperitoneal administration of antrocin significantly suppressed the growth of lung cancer tumor xenografts. Our results indicate that antrocin may be a potential therapeutic agent for human lung cancer cells through constitutive inhibition of JAK2/STAT3 pathway.

Uralsaponins M–Y, Antiviral Triterpenoid Saponins from the Roots of Glycyrrhiza uralensis

Thirteen new oleanane-type triterpenoid saponins, uralsaponins M-Y (1-13), and 15 known analogues (14-28) were isolated from the roots of Glycyrrhiza uralensis Fisch. The structures of 1-13 were identified on the basis of extensive NMR and MS data analyses. The sugar residues were identified by gas chromatography and ion chromatography coupled with pulsed amperometric detection after hydrolysis. Saponins containing a galacturonic acid (1-3) or xylose (5) residue are reported from Glycyrrhiza species for the first time. Compounds 1, 7, 8, and 24 exhibited good inhibitory activities against the influenza virus A/WSN/33 (H1N1) in MDCK cells with IC50 values of 48.0, 42.7, 39.6, and 49.1 μM, respectively, versus 45.6 μM of the positive control oseltamivir phosphate. In addition, compounds 24 and 28 showed anti-HIV activities with IC50 values of 29.5 and 41.7 μM, respectively.

Identification of Key Licorice Constituents Which Interact with Cytochrome P450: Evaluation by LC/MS/MS Cocktail Assay and Metabolic Profiling

Licorice has been shown to affect the activities of several cytochrome P450 enzymes. This study aims to identify the key constituents in licorice which may affect these activities. Bioactivity assay was combined with metabolic profiling to identify these compounds in several complex licorice extracts. Firstly, the inhibition potencies of 40 pure licorice compounds were tested using an liquid chromatography/tandem mass spectrometry cocktail method. Significant inhibitors of human P450 isozymes 1A2, 2C9, 2C19, 2D6, and 3A4 were then selected for examination of their structural features by molecular docking to determine their molecular interaction with several P450 isozymes. Based on the present in vitro inhibition findings, along with our previous in vivo metabolic studies and the prevalence of individual compounds in licorice extract, we identified several licorice constituents, viz., liquiritigenin, isoliquiritigenin, together with seven isoprenylated flavonoids and arylcoumarins, which could be key components responsible for the herb–drug interaction between cytochrome P450 and licorice. In addition, hydrophilic flavonoid glycosides and saponins may be converted into these P450 inhibitors in vivo. These studies represent a comprehensive examination of the potential effects of licorice components on the metabolic activities of P450 enzymes.

Separation and detection of minor constituents in herbal medicines using a combination of heart-cutting and comprehensive two-dimensional liquid chromatography.

Herbal medicines contain a large number of minor constituents, which could contribute to their therapeutic effects and provide valuable lead compounds for drug discovery. However, to explore minor constituents from complicated herbal extracts is usually laborious and time-consuming. In order to discover minor novel herbal constituents efficiently, we combined heart-cutting and comprehensive two-dimensional liquid chromatography (HC-2DLC) to remove major components from herbal extracts, and then characterized the minor ones by mass spectrometry. This strategy was employed to analyze Pueraria lobata and Pueraria thomsonii, the roots of which are used as the Chinese herbal medicine Ge-Gen. Five major compounds in Ge-Gen extract were removed by on-line heart-cutting, and the minor compounds were separated on an RP×RP 2DLC system (1D, Acquity CSH C18, 2.1×100mm, 1.7μm; 2D, Poroshell Phenyl-Hexyl, 3.0×50mm, 2.7μm). A synchronized gradient elution program was used to improve chromatographic resolution of the second dimension. By using this 2DLC system, a total of 271 and 254 peaks were separated in P. lobata and P. thomsonii within 35min, respectively. The practical and effective peak capacity was 1593 and 677, respectively, and the orthogonality was around 70%. Structures of 12 selected compounds were tentatively characterized by mass spectrometry, and 9 of them were discovered from Ge-Gen for the first time. Contents of these minor compounds in Ge-Gen were preliminarily determined to be 0.01-0.1% (w/w). The HC-2DLC/MS system is a powerful and convenient tool to explore minor novel chemical constituents from complex herbal extracts.

Separation of 25R/S-ergostane triterpenoids in the medicinal mushroom Antrodia camphorata using analytical supercritical-fluid chromatography

Ergostanes are major bioactive constituents of the medicinal mushroom Antrodia camphorata. These tetracyclic triterpenoids usually occur as 25R/S epimeric pairs, which renders their chromatographic separation difficult. In this study, we used analytical supercritical-fluid chromatography (SFC) to separate seven pairs of 25R/S-ergostanes from A. camphorata. The (R)- and (S)-forms for each of the seven pairs could be well resolved (Rs>1.3) on a Chiralcel OJ-H column (4.6×250mm, 5μm, chiral), eluted by 10% MeOH in CO2 at 2mL/min with a back pressure of 120bar and a column temperature of 40°C. Particularly, this chiral-SFC method could rapidly and efficiently separate low-polarity epimers like antcin A and antcin B, which were very difficult for RP-HPLC. A 3-min preparative-scale method was established to purify (25S)- and (25R)-antcin A for the first time. However, OJ-H column suffered from peak overlapping of different pairs of ergostanes. We found that Princeton 2-ethylpyridine column (2-EP, 4.6×250mm, 3μm, achiral) could effectively separate different pairs, although the resolutions for 25-R/S forms of each epimeric pair were not as good as OJ-H column. Meanwhile, all the (25S)-forms showed stronger retentions than the corresponding (25R)-epimers on the 2-EP column. These results demonstrated different selectivity of chiral- and achiral-SFC in separating 25R/S-ergostane epimers. Aside from high separation efficiency, SFC also showed advantage over HPLC in short analysis time and low consumption of organic solvents. Finally, both OJ-H and 2-EP columns were used on analytical SFC to separate 25R/S-ergostanes in an extract of A. camphorata.

Antcamphins A–L, Ergostanoids from Antrodia camphorata

Twelve ergostanoids, named antcamphins A-L (1-12), together with 20 known triterpenoids, were isolated from fruiting bodies of the medicinal fungus Antrodia camphorata. Compounds 1 and 2 represent the first examples of norergostanes isolated from A. camphorata, and compounds 3 and 4 are the first pair of cis-trans isomers of ergostane-type triterpenoids containing an aldehyde group. Compounds 5-12 are four pairs of C-25 epimers. The structures of 1-12 were elucidated on the basis of extensive spectroscopic data analysis including NMR and HRESIMS. Particularly, the absolute configurations at C-25 for 5-12 were determined by the modified Moshers method. These triterpenoids exhibited weak cytotoxic activities against MDA-MB-231 breast cancer cells and A549 lung cancer cells, but did not inhibit the growth of normal cells in the sulforhodamine B assay.