Yan-Yu Lan

Rapid screening and identification of caffeic acid and its esters in Erigeron breviscapus by ultra-performance liquid chromatography/tandem mass spectrometry

Caffeic acid and its esters (CAEs) are widely distributed in the plant kingdom and have been reported to elicit a wide range of exceptional biological activities. Present methods for screening and characterization of CAEs normally need the use of liquid chromatography diode-array detection/multistage mass spectrometry (LC-DAD/MS(n)). In this report, a rapid and efficient method coupling ultra-performance liquid chromatography (UPLC) with fragment-targeted multi-reaction monitoring (MRM) has been developed for screening CAEs in a crude extract of Erigeron breviscapus, while a UPLC/quasi-MS(n) method has been applied in the structural identification of these compounds. Furthermore, a simple quasi-UPLC/MS/MS method based on in-source collision-induced dissociation (CID) has been proposed for rapid identification of the CAEs. As a result, a total of more than 34 CAEs were detected and their structures characterized. Nine of them were reported from E. breviscapus for the first time. Applications of these strategies in the chemical investigation of an injection of E. breviscapus resulted in the identifications of 16 CAEs. These strategies, if appropriate modifications are made, will be very useful in screening and characterization not only of CAEs, but of other structural types of compounds in various complex matrices.

Antibacterial and anti-inflammatory effects of extracts and fractions from Polygonum capitatum

AIM OF THE STUDY This study was designed to investigate the antibacterial and anti-inflammatory activities of the extracts and the structure-based fractions from P. capitatum so as to provide the evidence for the traditional use of this plant in the treatment of urinary tract infections and to clarify the structural types that were responsible for the clinical use of the plant. MATERIALS AND METHODS The dry whole plant of P. capitatum was extracted with water and 70% aqueous ethanol and then separated, respectively, into a fraction enriched in polysaccharides and proteins (PP) and four other fractions enriched in gallic acid and its analogues (GAA), flavonoids (FV), tannins (TN), and triterpenoids and steroids (TS). UV spectral or chemical methods were used for the confirmation of the five fractions. The in vitro antibacterial activities of the aqueous (AE) and 70% aqueous ethanol (70EE) extracts as well as the fractions against gram-positive and gram-negative bacteria were initially evaluated by a disc diffusion test. The anti-bacterial potencies of the active extracts or fractions were then assessed in vitro by determining the MICs and MBCs. The anti-inflammatory activity was evaluated employing the xylene-induced mouse ear edema model. RESULTS Except for fraction PP, AE, 70EE, and the four fractions (GAA, FV, TN, and TS) exhibited varying degrees of antibacterial and anti-inflammatory activities. The results of the minimal inhibition concentration (MIC) and minimal bactericidal concentration (MBC) indicated that the crude extracts or fractions FV and TN all possess bacteriostatic and bactericidal properties. Fractions FV and TS showed significantly anti-inflammatory activity (P<0.01) with the inhibition rates of 86.15 and 73.71% at 0.6g/kg, respectively, as compared to 76.93% of the positive control dexamethasone. CONCLUSIONS The overall results suggested that the traditional use of this plant for the treatment of urinary tract infections were attributed to the presence of antibacterial and anti-inflammatory agents. The results also provided evidence that the studied plant extracts, as well as some of the fractions obtained from this plant might be potential sources for antimicrobial and anti-inflammatory drug development.

A UPLC-MS/MS method for simultaneous determination of danshensu, protocatechuic aldehyde, rosmarinic acid, and ligustrazine in rat plasma, and its application to pharmacokinetic studies of Shenxiong glucose injection in rats.

A rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the simultaneous determination of the four major active ingredients, danshensu, protocatechuic aldehyde, rosmarinic acid, and ligustrazine, in the traditional Chinese medicine Shenxiong glucose injection in rat plasma. Acidified and alkalized plasma samples were extracted using ethyl acetate, and separated on a Waters C18 column (2.1mm×50mm, 1.7μm) by using a gradient mobile phase system of acetonitrile-water containing 0.1% formic acid and luteoloside as an internal standard. Electrospray ionization in the positive-ion mode and multiple reaction monitoring were used to identify and quantitate the active components. All calibration curves showed good linearity (r>0.994) over the concentration range, with a lower limit of quantification (LLOQ) between 0.02 and 0.21μg/mL. The precision of the in vivo study was evaluated by intra- and inter-day assays, and the percentage of relative standard deviation was within 15%. Moreover, satisfactory extraction efficiency was obtained (between 83.94 and 117.81%) by liquid-liquid extraction. The validated method was successfully applied in a pharmacokinetic study in rats after intravenous administration of Shenxiong glucose injection. The results showed that the four bioactive ingredients in Shenxiong glucose injection have linear pharmacokinetic properties in rats after intravenous injection within the administered dose range and partially different ones compared to single ingredient.

Identification and Characterisation of Phenolics in Polygonum capitatum by Ultrahigh‐Performance Liquid Chromatography with Photodiode Array Detection and Tandem Mass Spectrometry

INTRODUCTION Polygonum capitatum is a well-known Chinese medicinal plant widely used by the Miao people for the treatment of various urologic disorders. Previous investigations have shown the presence of various types of phenolics. Our ultrahigh-performance liquid chromatography with photodiode array detection and mass spectrometry (UPLC-PDA-MS) analysis indicated that flavonoid glycosides and polyphenolic glycosides were its major constituents and quite a number of phenolic compounds have not yet been identified. Identification or characterisation of the major compounds of this plant will contribute to the scientific understanding of the medicinal plant and the authentication of the plant material and its pharmaceutical preparations. OBJECTIVE To develop an efficient method for the identification and structural characterisation of the major compounds present in P. capitatum. METHODS Elution of the 70% ethanol extract of P. capitatum by 80% ethanol on a D101 macroporous resin column afforded a phenolics-enriched fraction, separation of which by Sephadex LH-20 column chromatography eluted with absolute ethanol gave a tannin-free phenolic fraction (TFPF). Compounds present in TFPF were identified and structurally characterised by UPLC-PDA-ESI-MS/MS. RESULTS An amino acid and 40 phenolics including a number of flavonoid glycosides and polyphenolic glycosides were identified or structurally characterised in TFPF. Among these compounds, four were new and 19 were described in the plant for the first time. CONCLUSION The study showed that TFPF of P. capitatum contained flavonoid glycosides and polyphenolic glycosides as its major principles. Polyphenolic glycosides were perhaps the most typical chemical markers of P. capitatum.

UPLC-PDA-ESI-MS/MS Analysis of Compounds Extracted by Cardiac h9c2 Cell from Polygonum orientale.

INTRODUCTION A flavonoid-enriched extract (FEE) of Polygonum orientale was reported to show cardioprotective effect but only very few compounds were reported to contribute to the effect. Identification of compounds interacting with the target cardiac cell is important for the understanding of active compounds. OBJECTIVE To develop an efficient method for the screening of potential active compounds directly acting on the target cardiac cell in FEE and to structurally characterise these compounds. METHODOLOGY Flavonoid-enriched extract was prepared by extraction of the plant with water, addition of ethanol to the solution to remove polysaccharides and proteins, and removal of tannins by a polyamide column chromatography. Cell extraction was conducted on a cardiac h9c2 cell and the solution containing compounds released from the cell were desalted by solid phase extraction. Compounds present in the cell extract were detected by ultra-performance liquid chromatography (UPLC) and targeted multi-reaction monitoring (MRM), while their structures were characterised by UPLC-photodiodide array (PDA)-electrospray ion source (ESI)-MS/MS investigations of the FEE. RESULTS Twenty-three potentially active phenolics including ten flavonoid C-glycosides and six flavonoid O-glycosides have been identified from the 40 compounds screened in the cell extract. Among these compounds, three were new and nine were identified from this plant for the first time. Strategies for the structural characterisation of flavonoid glycosides were also discussed. CONCLUSION The study has shown that FEE contains the flavonoid as its major principles and the coupling of UPLC-PDA-ESI-MS/MS and targeted UPLC-MRM with target cell extraction is an efficient method for the screening and structural characterisation of potential active compounds.

Protective effects of fractions from Pseudostellaria heterophylla against cobalt chloride-induced hypoxic injury in H9c2 cell

ETHNOPHARMACOLOGICAL RELEVANCE Except for as a well-known tonic Chinese herbal medicine for the treatment of splenic asthenia, anorexia, lassitude and weakness, the roots of Pseudostellaria heterophylla was also used in Chinese medicines for the treatment of palpitation. AIM OF THE STUDY The study was designed to determine whether fractions from Pseudostellaria heterophylla could provide cardioprotection on hypoxic cardiomyocytes, what structural types of compounds were responsible for the observed effects, and which is the possible mechanism of action. MATERIALS AND METHODS The roots of Pseudostellaria heterophylla were extracted successively with 70% aqueous ethanol and water to give a 70% ethanol extract and a water extract. The latter was first precipitated by 80% ethanol and then protein-removed by the Sevag method to give a fraction enriched in polysaccarides (PHP). The former was separated by column chromatography into a fraction enriched in small-molecule sugars and amino acids (PHSSAC), saponins (PHS), cyclopeptides (PHCP), and sapogenins (PHSG). UV spectral or chemical methods were used to confirm the five fractions. The cardioprotective effects of the fractions were evaluated by measuring the viability and the leakage of lactate dehydrogenase (LDH) of the fraction-pretreated cardiomyocyte H9c2 after exposure to CoCl2-induced hypoxia. The mechanism of action was studied by investigating the nature of cell death inhibition (by Annexin V/PI flow cytometric analysis) and their effects on the levels of malonaldehyde (MDA), superoxide dismutase (SOD) and intracellular reactive oxygen species (ROS). RESULTS Fractions PHS and PHP could attenuate CoCl2-induced hypoxic damage to an extent higher than or comparable to the effect of the positive control N-acetyl-l-cysteine (NAC). Pretreatment of the cells with 800 μg/mL of PHS or 10mg/mL of PHP markedly decreased the level of MDA, reduced intracellular ROS, increased the activity of SOD, and reduced leakage of LDH to the levels close to or better than that with 326 μg/mL of NAC. Reduction of apoptosis was also observed for both fractions. CONCLUSIONS The overall results suggested that the traditional use of this plant for the treatment of palpitation may be attributed to the presence of cardioprotective agents in Pseudostellaria heterophylla. PHP and PHS were the two active fractions responsible for its cardioprotective effect. The mechanism might involve protections of the cell membrane from hypoxic damage and of the cells from oxidative injury via preventing increased oxidative stress. Protection of the cells via inhibition of cellular apoptosis may also be involved.

Electrospray ionization and collision-induced dissociation tandem mass spectrometric discrimination of polyphenolic glycosides: exact acylation site determination of the O-acylated monosaccharide residues.

RATIONALE Acylated monosaccharide residues are structural subunits of natural products or synthetic intermediates that have received much attention in past years. Determination of the acylation sites of these residues still relies heavily on the comparison of their characteristic NMR signals with those of known standards and synthesized acylated glycosides. It is important to develop a rapid analytical method for determining the acylation sites for these compounds, and this is described in this study. METHODS Six known polyphenolic glycosides were used for the electrospray ionization and collision-induced dissociation tandem mass spectrometry (ESI-CID-MS/MS) discrimination of the acylated monosaccharide residues with different acylation sites. A combination of ESI-CID-MS/MS, using a triple quadrupole mass spectrometer, with ultra-performance liquid chromatography (UPLC) and photo-diode array (PDA) detection (UPLC-PDA) has been applied to the identification or characterization of polyphenolic glycosides in Polygonum capitatum that possess an acylated monosaccharide residue. RESULTS An ESI-MS and CID-MS/MS method has been developed for the determination of the acylation sites of polyphenolic glycosides that possess an acylated monosaccharide residue. Twelve polyphenolic glycosides including four new ones have been identified or characterized in P. capitatum. Eight (including the new ones) of the twelve glycosides were reported for the first time from this plant. CONCLUSIONS The developed ESI-MS and CID-MS/MS method provided a very useful strategy for the determination of the sites of polyphenolic glycosides that possess an acylated monosaccharide residue. The acylation site could be determined by the characteristic product ion spectra of the in-source CID-generated O-acyl monosaccharide ion [B(1)](+). The presented work may facilitate the structural characterization of these types of compounds.

Two new phenolic glycosides from Inula cappa

Two new phenolic glycosides, syringic acid-4-O-α-L-rhamnoside (1) and ( − )-hydnocarpin-7-O-β-D-glucoside (2), were isolated from the traditional Chinese medicinal herb Inula cappa. The structures of the new compounds were elucidated by means of spectroscopic methods such as 1D, 2D NMR, and HR-ESI-MS.

Simultaneous determination of human plasma protein binding of bioactive flavonoids in Polygonum orientale by equilibrium dialysis combined with UPLC–MS/MS

A simple and selective ultra performance liquid chromatography–electrospray ionization tandem mass spectrometry (UPLC–ESI-MS/MS) assay was developed for the determination of the human plasma protein binding of four bioactive flavonoids (such as orientin and vitexin) in Polygonum orientale. Protein precipitation was used for sample preparation. Equilibrium dialysis technique was applied to determine the plasma protein binding under physiological conditions. The separation was achieved through a Waters C18 column with a mobile phase composed of 0.1% formic acid in acetonitrile and 0.1% aqueous formic acid using step gradient elution at a flow rate of 0.35 mL/min. A Waters ACQUITY™ TQD system was operated under the multiple reaction monitoring (MRM) mode of positive electrospray ionization. All of the recovery, precision, accuracy and stability of the method met the requirements. Good correlations (r>0.99) of the four compounds were found, which suggested that these compounds can be simultaneously determined with acceptable accuracy. Results showed that the plasma protein bindings of the four bioactive flavonoids were in the range of 74–89% over the six concentrations studied. The binding parameters containing protein binding affinity, protein binding dissociation constant, and protein binding site were studied. The maximum ability to bind with protein was also determined in the assay in order to understand the drug-protein binding of each compound better.

Synthesis, anti-HBV activity and renal cell toxicity evaluation of mixed phosphonate prodrugs of adefovir

A series of phosphonate ester prodrugs of adefovir incorporating l-amino (thio)acid and non-steroidal anti-inflammatory drug (NSAID) moieties were synthesized and their anti-HBV activity and renal cell toxicity were evaluated in HepG2 2.2.15 and HK-2 cells respectively. Bioactivity evaluation results revealed that this kind of adefovir prodrug have lower renal cell toxicity than adefovir dipivoxil. Compounds 8a and 8b, incorporating the NSAID ketoprofen and the l-amino acid (Val or Ile) structural fragments, exhibited more potent anti-HBV activity than adefovir dipivoxil with IC(50) = 0.51 and 0.73 μM, SI = 1697.64 and 881.92 respectively. In vitro stability studies showed that the synthesized prodrugs have higher chemical and plasma stability than the positive control adefovir dipivoxil.