Shuai Ji

Bioactive Constituents of Glycyrrhiza uralensis (Licorice): Discovery of the Effective Components of a Traditional Herbal Medicine.

Traditional herbal medicines have been reported to possess significant bioactivities. In this investigation, a combined strategy using both phytochemical and biological approaches was conducted to discern the effective components of licorice, a widely used herbal medicine. Altogether, 122 compounds (1-122), including six new structures (1-6), were isolated and identified from the roots and rhizomes of Glycyrrhiza uralensis (licorice). These compounds were then screened using 11 cell- and enzyme-based bioassay methods, including Nrf2 activation, NO inhibition, NF-κB inhibition, H1N1 virus inhibition, cytotoxicity for cancer cells (HepG2, SW480, A549, MCF7), PTP1B inhibition, tyrosinase inhibition, and AChE inhibition. A number of bioactive compounds, particularly isoprenylated phenolics, were found for the first time. Echinatin (7), a potent Nrf2 activator, was selected as an example for further biological work. It attenuated CCl4-induced liver damage in mice (5 or 10 mg/kg, ip) and thus is responsible, at least in part, for the hepatoprotective activity of licorice.

Rapid chemical profiling of saponins in the flower buds of Panax notoginseng by integrating MCI gel column chromatography and liquid chromatography/mass spectrometry analysis.

The flower buds of Panax notoginseng (Notoginseng flower, FBP) are used as the traditional Chinese medicine San-Qi-Hua. In this study, we conducted column chromatography fractionation and liquid chromatography/mass spectrometry (LC/MS) analysis to comprehensively profile bioactive notoginseng saponins (ginsenosides) in FBP. MCI gel column chromatography allowed separation and enrichment of minor saponins. Electrospray ionization tandem mass spectrometry of [M-H](-) and [M+Na](+) precursor ions of the saponins provided reliable structural information for the sapogenin, and sequence of sugar chains. Confirmed by high-accuracy Q-TOF analysis, 170 notoginseng saponins were characterized from FBP, and 91 of them were reported from Panax species for the first time. The new ginsenosides contain acyl groups on α-chain, malonyl group at 20-OH, or di-malonyl groups. This study also indicated that the flower buds of P. notoginseng contained more protopanaxadiol-type but less protopanaxatriol-type ginsenosides than the roots.

New triterpene saponins from the roots of Glycyrrhiza yunnanensis and their rapid screening by LC/MS/MS

The roots of Glycyrrhiza yunnanensis Cheng f. et L. K. Dai ex P. C. Li are used as licorice in traditional medicine of Southwest China. Triterpene saponins are the major chemical constituents. In this study, one new oleanane-type triterpenoid, glyyunnansapogenin I (I), seven new triterpene saponins, yunganosides E3 (II), L (III), M (IV), N1 (V), O (VI), P (VII) and N2 (VIII), together with four known saponins (IX-XII) were isolated from the roots of G. yunnanensis by preparative chromatography. Their structures were identified by spectroscopic analysis including NMR and HR-MS. Based on (-)-ESI-MS/MS fragmentation behaviors of these reference standards, an LC/MS/MS method using neutral loss scan and precursor ion scan on a triple quadrupole mass spectrometer was established to rapidly and comprehensively analyze triterpene saponins in G. yunnanensis. Combined with high-accuracy qTOF mass spectrometry analysis, a total of 50 saponins were detected, and their structures were identified or tentatively characterized. This is the first systematic study on triterpene saponins in G. yunnanensis.

Global Profiling and Novel Structure Discovery Using Multiple Neutral Loss/Precursor Ion Scanning Combined with Substructure Recognition and Statistical Analysis (MNPSS): Characterization of Terpene-Conjugated Curcuminoids in Curcuma longa as a Case Study

To fully understand the chemical diversity of an herbal medicine is challenging. In this work, we describe a new approach to globally profile and discover novel compounds from an herbal extract using multiple neutral loss/precursor ion scanning combined with substructure recognition and statistical analysis. Turmeric (the rhizomes of Curcuma longa L.) was used as an example. This approach consists of three steps: (i) multiple neutral loss/precursor ion scanning to obtain substructure information; (ii) targeted identification of new compounds by extracted ion current and substructure recognition; and (iii) untargeted identification using total ion current and multivariate statistical analysis to discover novel structures. Using this approach, 846 terpecurcumins (terpene-conjugated curcuminoids) were discovered from turmeric, including a number of potentially novel compounds. Furthermore, two unprecedented compounds (terpecurcumins X and Y) were purified, and their structures were identified by NMR spectroscopy. This study extended the application of mass spectrometry to global profiling of natural products in herbal medicines and could help chemists to rapidly discover novel compounds from a complex matrix.

Separation and characterization of phenolic compounds and triterpenoid saponins in licorice (Glycyrrhiza uralensis) using mobile phase-dependent reversed-phase×reversed-phase comprehensive two-dimensional liquid chromatography coupled with mass spectrometry.

Licorice is one of the most popular herbal medicines worldwide. It contains a big array of phenolic compounds (flavonoids, coumarins, and diphenylethanones). Due to high structural diversity, low abundance, and co-elution with licorice saponins, these phenolic compounds are difficult to be separated by conventional chromatography. In this study, a mobile phase-dependent reversed-phase×reversed phase comprehensive two-dimensional liquid chromatography (RP×RP 2DLC) method was established to separate phenolic compounds in licorice (the roots of Glycyrrhiza uralensis). Organic solvents in the mobile phase were optimized to improve orthogonality of the first and second dimensions, and a synchronized gradient mode was used to improve chromatographic resolution. Finally, licorice extracts were eluted with methanol/water/formic acid in the first dimension (Acquity CSH C18 column), and acetonitrile/water/formic acid in the second dimension (Poroshell Phenyl-Hexyl column). By using this 2DLC system, a total of 311 compounds were detected within 40min. The practical and effective peak capacity was 1329 and 524, respectively, and the orthogonality was 79.8%. The structures of 21 selected unknown compounds were tentatively characterized by mass spectrometry, and 8 of them were discovered from G. uralensis for the first time. The mobile phase-dependent 2DLC/MS system could benefit the separation and characterization of natural products in complicated herbal extracts.

Terpecurcumins A–I from the Rhizomes of Curcuma longa: Absolute Configuration and Cytotoxic Activity

Terpecurcumins A-I (1-9), together with three known analogues (10-12), were isolated from the rhizomes of Curcuma longa (turmeric). They were derived from the hybridization of curcuminoids and bisabolanes. The structures and absolute configurations of 1-9 were elucidated on the basis of extensive spectroscopic data analysis, including NMR and electronic circular dichroism spectra. The configuration of 10 was further confirmed by X-ray crystallography. A plausible biogenetic relationship for 1-12 is proposed. Compounds 4, 6, 7, 10, and 11 showed higher cytotoxic activities (IC(50), 10.3-19.4 μM) than curcumin (IC(50), 31.3-49.2 μM) against human cancer cell lines (A549, HepG2, and MDA-MB-231).

Anti-H1N1 virus, cytotoxic and Nrf2 activation activities of chemical constituents from Scutellaria baicalensis.

ETHNOPHARMACOLOGICAL RELEVANCE Huang-Qin, derived from the roots of Scutellaria baicalensis Georgi, is a popular Chinese herbal medicine mainly used to treat influenza and cancer. This study aims to elucidate the anti-influenza, anti-cancer and anti-oxidation effective components of S. baicalensis. MATERIALS AND METHODS Various column chromatography techniques and semi-preparative HPLC were used to isolate Scutellaria compounds, and their structures were identified by HRESIMS and NMR spectroscopic analysis. The pure compounds were evaluated for anti-influenza activities against A/WSN/33 (H1N1) virus in MDCK cells, cytotoxic activities against HepG2, SW480 and MCF7 human cancer cells by MTS assay, and antioxidant activities by Nrf2 luciferase reporter assay. In addition, the contents of 12 major compounds in 27 batches of S. baicalensis were simultaneously determined by a fully validated UPLC/UV method. RESULTS A total of thirty compounds (1-30), including four new ones (3, 7, 11 and 23), were isolated from S. baicalensis. Baicalin (15), baicalein (26), wogonin (27), chrysin (28) and oroxylin A (30) showed potent anti-H1N1 activities, with IC50 values of 7.4, 7.5, 2.1, 7.7 and 12.8 μM, respectively, which were remarkably more potent than the positive drug Osv-P (oseltamivir phosphate, IC50 45.6 μM). Most free flavones (26-28 and 30) showed significant cytotoxic activities at 10 μM (up to 61.2% inhibition rate). Furthermore, 30 could activate Nrf2 transcription by 3.8-fold of the control at 10 μM. UPLC analysis indicated the 12 major compounds (including the bioactive ones) accounted for 195.93 ± 43.9 mg g(-)(1) of the herbal materials. CONCLUSION This study demonstrated that free flavones showed potent anti-influenza, anti-cancer and anti-oxidative activities. They are important effective components of S. baicalensis, and can be used as chemical markers for quality control of this herbal medicine.

Density Functional Theory Calculations in Stereochemical Determination of Terpecurcumins J–W, Cytotoxic Terpene-Conjugated Curcuminoids from Curcuma longa L.

Fourteen novel terpene-conjugated curcuminoids, terpecurcumins J-W (1-14), have been isolated from the rhizomes of Curcuma longa L. Among them, terpecurcumins J-Q and V represent four unprecedented skeletons featuring an unusual core of hydrobenzannulated[6,6]-spiroketal (1 and 2), bicyclo[2.2.2]octene (3-7), bicyclo[3.1.3]octene (8), and spiroepoxide (13), respectively. The structures of compounds 1-14 were elucidated by extensive spectroscopic analysis, and their absolute configurations were established by electronic circular dichroism, vibrational circular dichroism, and (13)C NMR spectroscopic data analysis, together with density functional theory calculations. The structure and configuration of 1 was further confirmed by single-crystal X-ray diffraction (Cu Kα). The biogenetic pathways of 1-14 were proposed, involving Michael addition, condensation, Diels-Alder cycloaddition, and electrophilic substitution reactions. Terpecurcumins showed more potent cytotoxic activities than curcumin and ar-/β-turmerone. Among them, terpecurcumin Q (8) exhibited IC50 of 3.9 μM against MCF-7 human breast cancer cells, and mitochondria-mediated apoptosis played an important role in the overall growth inhibition. Finally, LC/MS/MS quantitative analysis of five representative terpecurcumins indicated these novel compounds were present in C. longa at parts per million level.

Simultaneous quantification of 50 bioactive compounds of the traditional Chinese medicine formula Gegen-Qinlian decoction using ultra-high performance liquid chromatography coupled with tandem mass spectrometry.

The quality evaluation of patent drugs derived from traditional Chinese medicine (TCM) compound formulas has been challenging due to their complex chemical composition. In this study, we developed a solution to evaluate the quality of Gegen-Qinlian Decoction (GQD), an ancient four-herb TCM formula for the treatment of diarrhea and diabetes, together with its derived patent drugs by simultaneously quantifying 50 bioactive compounds. The samples were extracted by 100% methanol (for hydrophobic compounds) and 50% methanol in water (for hydrophilic compounds), respectively, and were separated on a Waters Acquity charged surface hybrid C18 column (2.1×100mm, 1.7μm) eluted with gradients of acetonitrile and water containing 0.1% formic acid at a flow rate of 400μL/min. The analytes were determined by ultra-high performance liquid chromatography coupled with tandem mass spectrometry in the selected reaction monitoring mode. The 50 compounds (including acidic and alkaline, hydrophilic and hydrophobic) were well resolved within 14min, and were determined using an internal standard method. The method was fully validated for precision, repeatability, and recovery. The limits of detection were 0.3-10.0ng/mL. Finally, this method was used to analyze 24 batches of GQD samples, including water decoction, pills, tablets, and oral solutions. Principal component analysis indicated significantly varied chemical compositions among these formulations. The tablets and pills contained higher concentrations of Scutellaria and Coptis compounds than the oral solutions, and the water decoction contained abundant glycosides and saponins. Moreover, the contents of flavanones and flavone O-glucuronides varied remarkably. This study provides a feasible solution for the comprehensive quality control of TCM patent drugs.

Efficient separation of curcumin, demethoxycurcumin, and bisdemethoxycurcumin from turmeric using supercritical fluid chromatography: From analytical to preparative scale.

Curcumin is the major constituent of turmeric (Curcuma longa L.). It has attracted widespread attention for its anticancer and anti-inflammatory activities. The separation of curcumin and its two close analogs, demethoxycurcumin and bisdemethoxycurcumin, has been challenging by conventional techniques. In this study, an environmentally friendly method based on supercritical fluid chromatography was established for the rapid and facile separation of the three curcuminoids directly from the methanol extract of turmeric. The method was first developed and optimized by ultra performance convergence chromatography, and was then scaled up to preparative supercritical fluid chromatography. Eluted with supercritical fluid CO2 containing 8-15% methanol (containing 10 mM oxalic acid) at a flow rate of 80 mL/min, curcumin, demethoxycurcumin and bisdemethoxycurcumin could be well separated on a Viridis BEH OBD column (Waters, 250 mm × 19 mm, 5 μm) within 6.5 min. As a result, 20.8 mg of curcumin (97.9% purity), 7.0 mg of demethoxycurcumin (91.1%), and 4.6 mg of bisdemethoxycurcumin (94.8%) were obtained after a single step of supercritical fluid chromatography separation with a mean recovery of 76.6%. Showing obvious advantages in low solvent consumption, large sample loading, and easy solvent removal, supercritical fluid chromatography was proved to be a superior technique for the efficient separation of natural products.