Gegen-Qinlian-Wan (GQW) is a popular traditional Chinese patent medicine for the treatment of diarrhea. It is composed of four herbal medicines, Puerariae Radix, Scutellariae Radix, Coptidis Rhizoma, and Glycyrrhizae Radix. In this study, a rapid and sensitive method based on ultra high-performance liquid chromatography coupled with diode-array detection and quadrupole time-of-flight mass spectrometry (UHPLC-DAD-qTOF-MS) was established to characterize the chemical constituents and rats metabolites of GQW. Samples were separated on an Agilent Zorbax Eclipse Plus-C18 column (100 mm × 2.1 mm, 1.8 μm) by gradient elution using acetonitrile and water containing 0.1% formic acid as the mobile phase. On the basis of UV and qTOF high-accuracy mass spectral analysis, a total of 62 compounds were identified or tentatively characterized from GQW, including 42 flavonoids, 8 alkaloids, 6 triterpenoids, 3 phenylethanoid glycosides, and 3 other types. Among them, 27 compounds were confirmed by comparing with reference standards. Furthermore, metabolites in rats plasma and urine after oral administration of GQW were also analyzed. A total of 42 compounds were identified, including 29 prototypes and 13 metabolites through metabolic pathways of demethylation, methylation, hydrolysis, sulfate conjugation, and glucuronide conjugation. Glucuronidated flavonoids were the main constituents in the plasma, and were then transformed into aglycones and excreted from urine. This is the first systematic study on the chemical constituents and metabolic profiling of GQW.

To discover new natural compounds from herbal medicines tends to be more and more difficult. In this paper, a strategy integrating orthogonal column chromatography and liquid chromatography/mass spectrometry (LC/MS) analysis was proposed, and was applied for rapid discovery of new ginsenosides from Panax ginseng (PG), Panax quinquefolium (PQ), and Panax notoginseng (PN). The ginsenosides extracts were fractionated by MCI gel × silica gel orthogonal column chromatography. The fractions were then separated on a C18 HPLC column, eluted with a three-component mobile phase (CH3CN/CH3OH/3 mM CH3COONH4H2O), and detected by electrospray ionization tandem mass spectrometry. The structures of unknown ginsenosides were elucidated by analyzing negative and positive ion mass spectra, which provided complementary information on the sapogenins and oligosaccharide chains, respectively. A total of 623 comprising 437 potential new ginsenosides were characterized from the ethanol extracts of PG, PQ and PN. New acylations, diversified saccharide chains and C-17 side chains constituted novelty of the newly identified ginsenosides. An interpretation guideline was proposed for structural characterization of unknown ginsenosides by LC/MS. To confirm reliability of this strategy, two targeted unknown trace ginsenosides were obtained in pure form by LC/MS-guided isolation. Based on extensive NMR spectroscopic analysis and other techniques, they were identified as 3-O-[6-O-(E)-butenoyl-β-d-glucopyranosyl(1,2)-β-d-glucopyranosyl]-20(S)-protopanaxadiol-20-O-β-d-glucopyranosyl(1,6)-β-d-glucopyranoside (named ginsenoside IV) and 3-O-β-d-glucopyranosyl(1,2)-β-d-glucopyranosyl-3β,12β,20(S),24(R)-tetra hydroxy-dammar-25-ene-20-O-β-d-glucopyranosyl(1,6)-β-d-glucopyranoside (ginsenoside V), respectively. The fully established structures were consistent with the MS-oriented structural elucidation. This study expanded our understanding on ginsenosides of Panax species, and the proposed strategy was proved efficient and reliable in the discovery of new minor compounds from herbal extracts.Graphical abstractHighlights► Orthogonal MCI and silica gel separation achieved enrichment of minor ginsenosides. ► LC–(±)ESI-MSn analysis offered complementary structure information of ginsenosides. ► 623 ginsenosides including 437 new ones were identified from three Panax species. ► LC/MS guided isolation of P. ginseng yielded two new ginsenosides. ► NMR analysis of two new ginsenosides was consistent with LC/MS analysis.

Although various techniques have been employed to analyze drug metabolites, the metabolism of multi-component herbal medicine has seldom been fully addressed. In contrast to chemical drugs, a number of compounds in herbal medicine could get into circulation and then be metabolized. Moreover, these compounds may have metabolic interactions which make their pharmacokinetics (PK) even more complicated. The present work aims to elucidate the multi-component pharmacokinetics of a herbal medicine, and to demonstrate how PK behaviors were altered by co-existing constituents. Licorice (Glycyrrhiza uralensis Fisch.), a most commonly used herbal medicine, was chosen as a model. A strategy was proposed to compare the PK profiles of licorice extract with those of nine single compounds. These compounds were major bioactive constituents of licorice, and represented various structural types (flavanone, chalcone, isoflavone, saponin, and coumarin). We established a segmented selected reaction monitoring LC/MS/MS method to simultaneously monitor 63 licorice metabolites in rat plasma, and obtained the PK profiles of 55 metabolites. The results indicated that interactions among licorice compounds altered their PK behaviors in 4 aspects: improvement in bioavailability for aglycones (133- and 109-fold increase for liquiritigenin and isoliquiritigenin, respectively), prolongation in system circulation for glycosides (0.3 h delay in Tmax for liquiritin apioside and isoliquiritin apioside), decrease of potential toxicity for saponins such as glycyrrhizic acid, and shift in plasma distribution for phase II metabolites. This is the first attempt to systematically reveal the in vivo process of licorice. Moreover, the study indicates noticeable interactions to alter pharmacokinetics among licorice compounds, which may be characteristic for herbal medicines.Highlights► PK of licorice extract and its constituents were studied comparatively. ► 63 licorice metabolites were monitored using a segmented LC/MS/MS method. ► The multi-component licorice extract altered the PK of single compounds. ► This is the first multi-component pharmacokinetic study of licorice.

Ingenol esters, one type of diterpenoids isolated from genus Euphorbia, were reported to possess cytotoxic activities. In our previous study, a number of ingenol esters were isolated from Euphorbia esula L. (Ru-Jiang-Da-Ji in Chinese). And as a part of the ongoing program, a study of fragmentation pathway of ingenol esters was carried out using mass spectrometry. The fragmentation behaviors of 17 standard compounds were investigated with ion trap (IT-MSn) mass spectrometry and hybrid quadrupole-time of flight-mass spectrometer (Q-TOF-MS/MS) in positive ion mode. Electrospray ionization (ESI) was used as ion source in analysis. According to the IT-MSn spectra, the elimination order of the acyloxy was R1OH > R2H > R3H > R4H, which was hard to determine in Q-TOF-MS/MS spectra. The Q-TOF-MS preferred to eliminate all the acyloxys along with some hydroxyls and produce three characteristic ions (x0, y0, z0), which indicate the numbers of the oxygenated groups. The Q-TOF-MS also provide high resolution mass spectra of the parent and product ions, which allowed the determination of the chemical formulas and characterization of the ion structures. These two instruments gave complementary information, which were very useful for structural characterization of the studied compounds.Graphical abstractHighlights► We studied the fragmentation pathways of ingenol esters firstly. ► Seventeen standards were provided. ► Both IT-MSn and Q-TOF-MS/MS were used. ► The IT-MSn determined the elimination order of the acyloxy groups. ► The Q-TOF-MS provided high resolution mass spectra of the parent and product ions.

Animal biles and gallstones are popularly used in traditional Chinese medicines, and bile acids are their major bioactive constituents. Some of these medicines, like cow-bezoar, are very expensive, and may be adulterated or even replaced by less expensive but similar species. Due to poor ultraviolet absorbance and structural similarity of bile acids, effective technology for species differentiation and quality control of bile-based Chinese medicines is still lacking. In this study, a rapid and reliable method was established for the simultaneous qualitative and quantitative analysis of 18 bile acids, including 6 free steroids (cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, hyodeoxycholic acid, and ursodeoxycholic acid) and their corresponding glycine conjugates and taurine conjugates, by using liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). This method was used to analyze six bile-based Chinese medicines: bear bile, cattle bile, pig bile, snake bile, cow-bezoar, and artificial cow-bezoar. Samples were separated on an Atlantis dC18 column and were eluted with methanol–acetonitrile–water containing ammonium acetate. The mass spectrometer was monitored in the negative electrospray ionization mode. Total ion currents of the samples were compared for species differentiation, and the contents of bile acids were determined by monitoring specific ion pairs in a selected reaction monitoring program. All 18 bile acids showed good linearity (r2 > 0.993) in a wide dynamic range of up to 2000-fold, using dehydrocholic acid as the internal standard. Different animal biles could be explicitly distinguished by their major characteristic bile acids: tauroursodeoxycholic acid and taurochenodeoxycholic acid for bear bile, glycocholic acid, cholic acid and taurocholic acid for cattle bile, glycohyodeoxycholic acid and glycochenodeoxycholic acid for pig bile, and taurocholic acid for snake bile. Furthermore, cattle bile, cow-bezoar, and artificial cow-bezoar could be differentiated by the existence of hyodeoxycholic acid and the ratio of cholic acid to deoxycholic acid. This study provided bile acid profiles of bile-based Chinese medicines for the first time, which could be used for their quality control.

A validated and sensitive HPLC–UV–MS method was developed for qualitative and quantitative analysis of curcuminoids in eight herbal medicines derived from four Curcuma species. The samples were separated on a YMC ODS-A C18 column with a gradient elution of acetonitrile and 0.1% formic acid. Curcumin, demethoxycurcumin and bisdemethoxycurcumin showed good linearity (r > 0.9998) in the concentration ranges of 4.88–625, 4.29–550 and 3.98–510 μg/mL, respectively. The results suggested that the contents of three major curcuminoids in different herbal medicines varied significantly. Curcuminoids were only detected in Jianghuang, HuangsiYujin, and PengEzhu. Amongst them, Jianghuang contained the highest amounts of curcuminoids (40.36 mg/g), which were almost 20 times higher than HuangsiYujin (1.94 mg/g) and 400 times higher than PengEzhu (0.098 mg/g). Furthermore, amongst the Jianghuang samples collected from different areas, samples from Sichuan Province contained remarkably higher amounts of curcuminoids (22.21–40.36 mg/g) than other cultivation regions.Research highlights► Curcuminoids are detected in Jianghuang, HuangsiYujin and PengEzhu. ► Curcuma longa (Jianghuang) contains highest amounts of curcuminoids. ► C. longa (Jianghuang) from Sichuan Province of China represents best quality.

This paper aims to investigate the metabolism and pharmacokinetics of curcumin, demethoxycurcumin and bisdemethoxycurcumin in mice tumor. To improve water solubility, nanoparticle formulations were prepared as curcuminoids-loaded solid lipid nanoparticles (curcuminoids-SLNs) and curcumin-loaded solid lipid nanoparticles (curcumin-SLNs). After intragastric administration to tumor-bearing ICR mice, the plasma and tumor samples were analyzed by liquid chromatography with ion trap mass spectrometry. We discovered that curcuminoids were mainly present as glucuronides in plasma, whereas in free form in tumor tissue. A validated LC/MS/MS method was established to determine the three free curcuminoids in tumor homogenate. Samples were separated on a Zorbax SB-C18 column, eluted with acetonitrile–water (containing 0.1% formic acid), and detected by TSQ Quantum triple quadrupole mass spectrometer in selected reaction monitoring mode. The method showed good linearity (r2 = 0.997–0.999) over wide dynamic ranges (2–6000 ng/mL). Variations within- and between-batch never exceeded 11.2% and 13.4%, respectively. The extraction recovery rates ranged from 78.3% to 87.7%. The pharmacokinetics of curcuminoids in mice tumor fit two-compartment model and first order elimination. For curcumin-SLNs group, the dosing of 250 mg/kg of curcumin resulted in AUC(0–48 h) of 2285 ng h/mL and Cmax of 209 ng/mL. For curcuminoids-SLNs group, the dosing equivalent to 138 mg/kg of curcumin resulted in higher tumor concentrations (AUC = 2811 ng h/mL, Cmax = 285 ng/mL). It appeared that co-existing curcuminoids improved the bioavailability of curcumin.

One monomer flavan-3-ol, 4α-carboxymethyl-(+)-catechin methyl ester, two monomer flavan-3-ol glycosides, (+)-afzelechin-3-O-β-allopyranoside, (+)-afzelechin-6-C-β-glucopyranoside, two dimer flavan-3-ols, (−)-epiafzelechin-(4β → 8)-4β-carboxymethyl-(−)-epicatechin methyl ester, and -(−)-epiafzelechin-(4β → 8)-4α-carboxymethyl-(−)epiafzelechin ethyl ester, and one trimer flavan-3-ol, (−)-epiafzelechin-(4β → 8)-(−)-epiafzelechin-(4β → 8)-4β-carboxymethyl-(−)-epiafzelechin methyl ester, together with thirteen known flavan-3-ols were isolated from the rhizomes of Drynaria fortunei (Kunze) J.Sm (Polypodiaceae). The structures were established by analysis of their HRESIMS, 1D, 2D NMR spectroscopic, and CD data. In order to obtain improved resolution, the high-resolution NMR spectra of the dimers and trimer were measured at −40 °C.Graphical abstractSix flavan-3-ols, including one monomer, two monomer glycosides, two dimers, and one trimer were isolated from the rhizomes of Drynaria fortunei (Polypodiaceae).Highlights► Six flavan-3-ols were isolated from Drynaria fortunei (Polypodiaceae). ► Low temperature NMR spectroscopy was used for identification of flavan-3-ol dimers and trimers. ► Aromatic hydrogen–deuterium exchange was observed during NMR determination of flavan-3-ols.

The biotransformation of three bioactive bufadienolides, namely, bufotalin (1), telocinobufagin (2), and gamabufotalin (3) by cell suspension cultures of Saussurea involucrata yielded 11 products. Bufotalin yielded 3-epi-bufotalin (1a), 3-epi-desacetylbufotalin (1b), 3-epi-bufotalin 3-O-β-d-glucoside (1c), 1β-hydroxybufotalin (1d), and 5β-hydroxybufotalin (1e); telocinobufagin yielded 3-dehydroscillarenin (2a), 3-dehydrobufalin (2b), and 3-epi-telocinobufagin (2c); and gamabufotalin yielded 3-epi-gamabufotalin (3a), 3-dehydrogamabufotalin (3b), and 3-dehydro-Δ1-gamabufotalin (3c), respectively. Among these 11 products, 1a, 1b, 1c, 1d, 3a and 3c are previously unreported. The structures of these metabolites were elucidated based on NMR spectroscopic analyses and mass spectrometry. Most metabolites showed significant cytotoxic activities against human hepatoma (HepG2) and breast cancer (MCF-7) cell lines. In addition, the time course for the biotransformation of 3 was investigated.Graphical abstractSix cytotoxic bufadienolides were obtained from biotransformation of bufotalin, telocinobufagin and gamabufotalin using cell suspension cultures of Saussurea involucrate. These compounds showed significant cytotoxic activities against human HepG2 hepatoma and MCF-7 breast cancer cells.Highlights► Six bufadienolides were obtained by biotransformation using cell suspension cultures of Saussurea involucrata. ► A Δ1-reduction was observed for the first time in the biotransformation of bufadienolides. ► The compounds showed potent cytotoxic activities against human hepatoma (HepG2) and breast cancer (MCF-7) cell lines with IC50 values of 0.05–0.50 μM.

A systematic study of the metabolites in Ganoderma lucidum led to isolation of 43 triterpenoids, six of them (1–6) are hitherto unknown. The structures of the latter were elucidated on the basis of spectroscopic studies and comparison with the known related compounds. All of the compounds were assayed for their inhibitory activities against human HeLa cervical cancer cell lines. Some compounds exhibit significant cytotoxicity, and their structure–activity relationships are discussed.Six triterpenoids, together with 37 known compounds, were isolated from Ganoderma lucidum. Their structures were elucidated on the basis of spectroscopic studies and comparison with known related compounds. All of the compounds were assayed for their inhibitory activity against human HeLa cervical cancer cell lines. In addition, structure–activity relationships are discussed.

The metabolites in rats after administration of icariside II, icariin, epimedin C and extracts of four Epimedium species were investigated. Feces, bile, plasma and urine samples were detected comprehensively using HPLC-ESI-MSn method. The structures of metabolites were identified on the basis of their characteristic fragmentations in MSn experiments. Totally, 54 metabolites were identified in these biosamples. Specific hydrolysis of 7-O glucosides in gut lumen and glucuronic acid conjugation in liver were considered as the main physiologic processes of prenylflavonoids. Icariside II and anhydroicaritin were the major intermediate products in forming of mono- and di-glucuronic acid conjugations in vivo. In general, this study revealed the possible metabolite profiles of prenylflavonoids in rats, and might aid the clinical use of different Epimedium species.

A pair of new flavanol racemates (1a and 1b) and a new flavanol racemic mixture (2) were isolated from crude propolis from Henan Province, People’s Republic of China. Also obtained were nine known compounds, including two flavones, four flavonols, two flavanols, and isoferulic acid. Spectroscopic analysis was employed to assign the structures of these new compounds and the absolute configurations of 1a and 1b. Cytotoxicity of the isolated compounds against the HeLa human cervical carcinoma cancer cell line was evaluated, with only compounds 1a, 1b, 2, and rhamnetin (3) being active.

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–MSn) 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.

Twelve new xanthones (1−12), a pair of new natural products (13 and 14), and 18 known related compounds were isolated from the resin of Garcinia hanburyi. The structures of 1−14 were elucidated by detailed spectroscopic analyses. A cytotoxic assay of the isolated compounds revealed that, with the exception of 2, these compounds were active against the HeLa tumor cell line.

Four new jatrophane-type diterpenoids (1−4), together with 16 known related compounds, were isolated from the Chinese medicinal plant Euphorbia helioscopia. The structures of 1−4 were determined on the basis of spectroscopic and chemical methods. Cytotoxicity of the isolated compounds against HeLa and MDA-MB-231 cells was evaluated, with only helioscopinolide A (5) and euphornin (3a) being active.

Two new isoprenylated flavanones, tonkinochromanes J (1) and K (2), and a new isoprenylated dihydrochalcone, tonkinochromane L (3), were isolated from the roots of Sophora tonkinensis along with four known compounds (4-7). Their structures were determined by means of spectroscopic analyses, including HRMS, IR, 1H and 13C NMR and 2D experiments (COSY, HSQC, and HMBC), and comparison with known related compounds.Two new isoprenylated flavanones, tonkinochromanes J (1) and K (2), and a new isoprenylated dihydrochalcone, tonkinochromane L (3), were isolated from the roots of Sophora tonkinensis along with four known compounds (4-7). Their structures were determined by means of spectroscopic analyses, including HRMS, IR, 1H and 13C NMR and 2D experiments (COSY, HSQC, and HMBC), and comparison with known related compounds.

A simple and sensitive high-performance liquid chromatographic method with diode-array detection has been developed and validated for determination of six bioactive constituents, baicalin (BG), wogonoside (WG), baicalein (BA), wogonin (WO), oroxylin A (OA), and rhein (RHE), in rat urine and bile after oral administration of Luan-Pao-Prescription. The separation was performed on a C18 column at 30 °C with a mobile phase gradient prepared from 0.5% aqueous glacial acetic acid and acetonitrile. Detection was at 320 nm for BG, WG, BA, WO, and OA, and 254 nm for RHE, for both biological fluids. Because of the properties of the compounds of interest in urine and bile, two methods of sample preparation were used—solid phase extraction for monitoring BG and WG and liquid–liquid extraction for BA, WO, OA, and RHE. The HPLC method was validated for linearity, LOD, LLOQ, precision, accuracy, and recovery from the biological fluids in accordance with ICH guidelines. The method was successfully used to investigate urinary and biliary excretion of the six constituents in rats.

Preparative-scale fermentation of ginsenoside Rb1 (1) with Acremonium strictum AS 3.2058 gave three new compounds, 12β-hydroxydammar-3-one-20 (S)-O-β-d-glucopyranoside (7), 12β, 25-dihydroxydammar-(E)-20(22)-ene-3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside (8), and 12β, 20 (R), 25-trihydroxydammar-3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside (9), along with five known compounds, ginsenoside Rd (2), gypenoside XVII (3), ginsenoside Rg3 (4), ginsenoside F2 (5), and compound K (6). The structural elucidation of these metabolites was based primarily on one- and two-dimensional nuclear magnetic resonance and high-resolution electron spray ionization mass spectra analyses. Among these compounds, 2–6 are also the metabolites of ginsenoside Rb1 in mammals. This result demonstrated that microbial culture parallels mammalian metabolism; therefore, A. strictum might be a useful tool for generating mammalian metabolites of related analogs of ginsenosides for complete structural identification and for further use in pharmaceutical research in this series of compounds. In addition, the biotransformation kinetics was also investigated.

An extensive study of metabolites present in Euphorbia esula led to isolation of 16 ingenane diterpenoids 1–16 together with the known ingenane derivative 17 and four known cycloartane triterpenoids. Their structures were elucidated on the basis of spectroscopic studies and comparison with known related compounds. All the compounds were assayed for their inhibitory activity against human HeLa cervical cancer cell line.Sixteen ingenane diterpenoids (1–16), together with five known compounds, were isolated from Euphorbia esula. Their structures were elucidated on the basis of spectroscopic studies and comparison with known related compounds. All the compounds were assayed for their inhibitory activity against human hela cervical cancer cell line.

High-performance liquid chromatographic (HPLC) fingerprints were developed for identification of both lipophilic and hydrophilic components of the roots of Salvia miltiorrhiza and four related preparations. These samples were separated with an Agilent Zorbax Extend C18 reserved-phase column (5 μm, 250 mm × 4.6 mm) by linear gradient elution using water-phosphoric acid (100:0.026, v/v) and acetonitrile as mobile phase. The flow rate was 0.8 ml/min and the detector wavelength was set at 280 nm. Mean chromatograms and correlation coefficients of samples were calculated by the software “Similarity Evaluation System for Chromatographic Fingerprint of TCM”. The correlation coefficients of Danshen and Fufang Danshen tablets (FDT) samples were in the range of 0.352–0.993 and 0.768–0.987, respectively. The correlation coefficients of Compound Danshen dripping pills (CDDP), Danshen injection (DSI) and Xiangdan injection (XDI) samples were higher than 0.928, 0.850 and 0.960, respectively. It was the first time to identify 34 peaks by comparing with standard compounds and using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MSn) technique. All results indicated that the developed fingerprint assay could be readily utilized as a quality control method for S. miltiorrhiza and its related preparations.

Triterpenoids extracted from Ganoderma lucidum (Leyss. ex Fr.) Karst were separated and characterized using optimized reversed-phase liquid chromatography with diode array detection and electrospray ion trap tandem mass spectrometry (HPLC-DAD-ESI-MSn). They could be classified into five types depending on the fragmentation behavior. All triterpenoids gave [M−H]− and [2M−H]− ions by electrospray ionization monitored in the negative ion mode; in addition, compounds of types III and IV gave prominent [M−H−H2O]− ions and the unsaturated bond at C-20, 22 would reduce the abundance of [M−H−H2O]− ion. The key fragmentation information was cleavage at C- and D-rings despite the predominant losses of H2O and CO2. Compounds with hydroxyls at C-7 and C-15 would produce a list of b, b−1, b−2, and b−16 ions attributed to cleavage of D-ring; if the second alcohol at C-15 were oxidized to ketone, the prominent cleavage would occur at C-ring and produce a group of ions of a; if C-7 were oxidized to ketone, transference of two hydrogen atoms would occur during the cleavage of rings and a list of ions about a+2 and/or b+2 would appear instead. The above fragmentations and regularities in fragmentation pathways were reported for the first time, and were implemented for the analysis of triterpenoids in G. lucidum. The chloroform extract was separated on a Zorbax SB-C18 column, eluting with an acetonitrile-0.2% acetic acid gradient. A total of 32 triterpenoids, including six new ones, were identified or tentatively characterized based on the tandem mass spectra of the HPLC peaks.

The major flavonoids in rat serum after oral administration of Dalbergia odorifera extract were analyzed qualitatively and quantitatively by high performance liquid chromatography (HPLC) and its coupling to mass spectrometry (HPLC–MS). Utilizing HPLC–MS technique, 18 flavonoids including five isoflavones, four isoflavanones, four neoflavones, two flavanones, two chalcones, one isoflavanonol were identified in free form in serum sample based on comparison with the authentic standards. Furthermore, the amounts of the four prominent flavonoids, (3R)-4′-methoxy-2′,3,7-trihydroxyisoflavanone, vestitone, formononetin and sativanone were determined in serum by HPLC–UV with internal standard method. The method was validated and utilized in pharmacokinetic studies of these four analytes. This is the first report on identification and determination of the major flavonoids in rat serum after oral administration of D. odorifera extract and the results provided a firm basis for clarifying the pharmacological effect of D. odorifera and evaluating the clinical applications of this medicinal herb.

Triterpenoids extracted from Ganoderma lucidum (Leyss. ex Fr.) Karst were separated and characterized using optimized reversed-phase liquid chromatography with diode array detection and electrospray ion trap tandem mass spectrometry (HPLC-DAD-ESI-MSn). They could be classified into five types depending on the fragmentation behavior. All triterpenoids gave [M – H]– and [2M – H]– ions by electrospray ionization monitored in the negative ion mode; in addition, compounds of types III and IV gave prominent [M – H – H2O]– ions and the unsaturated bond at C-20, 22 would reduce the abundance of [M – H – H2O]– ion. The key fragmentation information was cleavage at C- and D-rings despite the predominant losses of H2O and CO2. Compounds with hydroxyls at C-7 and C-15 would produce a list of b, b – 1, b – 2, and b – 16 ions attributed to cleavage of D-ring; if the second alcohol at C-15 were oxidized to ketone, the prominent cleavage would occur at C-ring and produce a group of ions of a; if C-7 were oxidized to ketone, transference of two hydrogen atoms would occur during the cleavage of rings and a list of ions about a + 2 and/or b + 2 would appear instead. The above fragmentations and regularities in fragmentation pathways were reported for the first time, and were implemented for the analysis of triterpenoids in G. lucidum. The chloroform extract was separated on a Zorbax SB-C18 column, eluting with an acetonitrile-0.2% acetic acid gradient. A total of 32 triterpenoids, including six new ones, were identified or tentatively characterized based on the tandem mass spectra of the HPLC peaks.

Licorice is one of the most popular herbal medicines worldwide, and is mainly used to moderate the characteristics of other herbs in Traditional Chinese Medicine. It is hypothesized that licorice exerts this role by regulating systemic metabolism. Bile acids play a critical role in lipid digestion and cholesterol metabolism, and are sensitive biomarkers for hepatic function. In this study, the regulatory effects of licorice on bile acid metabonome in rats were investigated using liquid chromatography coupled with tandem mass spectrometry. After oral administration of a clinical dosage of licorice water extract, the levels of 21 fully identified and 41 tentatively characterized bile acid analogs in rat plasma were determined by a fully validated method. Following partial least squares discriminant analysis, the results showed that licorice treatment led to dose-dependent up-regulation of free and glycine-conjugated bile acids excretion. Particularly, the plasma levels of cholic acid (1465.33 ± 915.93–7156.46 ± 3490.49 ng/mL, p = 0.0027) and β-muricholic acid (228.19 ± 163.95–1284.40 ± 775.62 ng/mL, p = 0.0045) increased significantly 48 h after administration. As licorice is widely used as a detoxifying drug, the regulation of plasma bile acids may be an important evidence to interpret its mechanism.Graphical abstractDownload full-size imageHighlights► Metabolic regulatory effect of licorice was studied through bile acid metabonomics. ► Sixty-two bile acid analogs were determined by LC-SRM/MS and analyzed by PLS-DA. ► Licorice intake led to dose-dependent up-regulation of free and glycine-bile acids. ► The results could contribute to interpret the detoxification mechanism of licorice.

To elucidate the cytotoxicity mechanism of Garnoderma triterpenes, a chemoproteomic study using five purified ganoderic acids, ganoderic acid F (GAF), ganoderic acid K (GAK), ganoderic B (GAB), ganoderic acid D (GAD) and ganoderic acid AM1 (GAAM1) was conducted. GAF, GAK, GAB, GAD and GAAM1 treatment for 48 h inhibited the proliferation of HeLa human cervical carcinoma cells with IC50 values of 19.5±0.6 μM, 15.1±0.5 μM, 20.3±0.4 μM, 17.3±0.3 μM, 19.8±0.7 μM, respectively. The protein expression profiles of HeLa cells treated with each ganoderic acid at dose of 15 μM for 48 h were checked using two-dimensional electrophoresis (2-DE). The possible target-related proteins of ganoderic acids, i.e. proteins with same change tendency in all five ganoderic acids-treated groups compared with control, were identified using MALDI-TOF MS/MS. Twelve proteins including human interleukin-17E, eukaryotic translation initiation factor 5A (eIF5A), peroxiredoxin 2, ubiquilin 2, Cu/Zn-superoxide dismutase, 14-3-3 beta/alpha, TPM4-ALK fusion oncoprotein type 2, PP2A subunit A PR65-alpha isoform, nucleobindin-1, heterogeneous nuclear ribonucleoprotein K, reticulocalbin 1 and chain A of DJ-1 protein were identified. Ganoderic acids might exert their cytotoxicity by altering proteins involved in cell proliferation and/or cell death, carcinogenosis, oxidative stress, calcium signaling and ER stress.

Bile acids are steroids with a pentanoic acid substituent at C-17. They are the terminal products of cholesterol excretion, and play critical physiological roles in human and animals. Bile acids are easy to detect but difficult to identify by using mass spectrometry due to their poly-ring structure and various hydroxylation patterns. In this study, fragmentation pathways of 18 free and conjugated bile acids were interpreted by using tandem mass spectrometry. The analyses were conducted on ion trap and triple quadrupole mass spectrometers. Upon collision-induced dissociation, the conjugated bile acids could cleave into glycine or taurine related fragments, together with the steroid skeleton. Fragmentations of free bile acids were further elucidated, especially by atmospheric pressure chemical ionization mass spectrometry in positive ion mode. Aside from universally observed neutral losses, eliminations occurred on bile acid carbon rings were proposed for the first time. Moreover, four isomeric 5β-cholanic acid hydroxyl derivatives (3α,6α-, 3α,7β-, 3α,7α-, and 3α,12α-) were differentiated using electrospray ionization in negative ion mode: 3α,7β-OH substituent inclined to eliminate H2O and CH2O2 groups; 3α,6α-OH substituent preferred neutral loss of two H2O molecules; 3α,12α-OH substituent apt to lose the carboxyl in the form of CO2 molecule; and 3α,7α-OH substituent exhibited no further fragmentation after dehydration. This study provided specific interpretation for mass spectra of bile acids. The results could contribute to bile acid analyses, especially in clinical assays and metabonomic studies.Graphical abstractDownload full-size imageHighlights► We studied the tandem mass spectrometry of 18 endogenous bile acids. ► Fragmentations on bile acid carbon rings were firstly interpreted by (+)-APCI MS. ► Isomeric bile acids were differentiated by (−)-ESI MS upon various collision energies.

Supercritical Fluid Extraction (SFE) technology has been used in Traditional Chinese Medicine (TCM), but some disadvantages still remain in the research and development of new drugs of TCM, especially when the drug is comprised of several plants. Through reviewing the R&D process of QinNao Capsules, the impacts on process and potential problems which may be caused in new drug R&D by SFE are discussed. Possible scenarios are proposed.

Salvianolic acids (SAs) extracted from Salvia miltiorrhiza could inhibit ADP-induced aggregation of rat washed platelets. To elucidate the cellular mechanism of the inhibitive effect on platelet aggregation, a proteomic method was used to find the possible target-related proteins of SA in platelets. Treatment of 150 μg/ml SA caused the regulation of platelet levels of 12 proteins, which play important roles in calcium homeostasis, antioxidant system and cytoskeleton structure. The proteomic result was also confirmed by Western blotting.We report the result of identifying the possible target-related proteins of salvianolic acids (SAs) in rat platelets. Twelve proteins that might be related to the inhibitive effect of salvianolic acids on platelet aggregation were identified.

Sanqi, the root of Panax notoginseng, is a popularly used traditional Chinese medicine with cardiovascular effects. Notoginsengnosides (NG) isolated from Sanqi could inhibit ADP-induced platelet aggregation of rat washed platelets. To identify the possible target proteins of NG in platelets, two-dimensional gel electrophoresis (2-DE)-based comparative proteomics was performed and proteins altered in expressional level after NG treatment were identified by MALDI-TOF MS/MS. Treatment of 200 μg/ml NG caused regulation of the levels of 12 proteins, which play important roles in platelet activation, oxidative stress and cytoskeleton. In the NG-treated platelets, there were increase in the levels of growth factor receptor-bound protein 2 (Grb2), thrombospondin 1, tubulin alpha 6 and decrease in the levels of thioredoxin, Cu–Zn superoxide dismutase, DJ-1 protein, peroxiredoxin 3, thioredoxin-like protein 2, ribonuclease inhibitor, potassium channel subfamily V member 2, myosin regulatory light chain 9 and laminin receptor 1. The change in the levels of these proteins caused by NG treatment might contribute to the inhibitive effect of NG on platelet aggregation. Furthermore, analysis of the reactive oxygen species (ROS) level indicated that NG could decrease the ROS level in platelets. The regulation of ROS level might play important role in the effect of NG on platelets.