Hui-Jun Li

Rapid and simple method for screening of natural antioxidants from Chinese herb Flos Lonicerae Japonicae by DPPH-HPLC-DAD-TOF/MS

A rapid and simple method has been developed for the screening and identification of natural antioxidants of Flos Lonicerae Japonicae (FLJ), derived from the flower buds of Lonicera japonica. The hypothesis is that upon reaction with 1,1-diphenyl-2-picrylhydrazyl (DPPH), the peak areas (PAs) of compounds with potential antioxidant effects in the HPLC chromatograms will be significantly reduced or disappeared, and the identity confirmation could be achieved by HPLC-DAD-TOF/MS hyphenated technique. Using the proposed approach, about 14 compounds in the FLJ extract were found to possess a potential antioxidant activity. They were identified as chlorogenic acid (1), 1-O-caffeoylquinic acid (1-O-CQA, 2), caffeic acid (4), 4-O-CQA (5), rutin (7), isoquercitrin (8), luteolin-7-O-glucoside (9), lonicerin (10), 4,5-O-dicaffeoylquinic acid (4,5-O-diCQA, 11), 3,5-O-diCQA (12), 1,3-O-diCQA (13), 3,4-O-diCQA (14), 1,4-O-diCQA (16), and luteolin (17). In addition, the free radical scavenging capacities of the available identified compounds were also investigated by HPLC assay. The results indicated that the compounds with PAs significantly decreasing were natural antioxidants, whereas those with PAs not changing presented no activities, which accordingly indicated that this newly proposed method could be widely applied for rapid screening and identification of natural antioxidants from complex matrices including Chinese herbal medicines.

Chemistry, bioactivity and geographical diversity of steroidal alkaloids from the Liliaceae family

Plants belonging to the Liliaceae family have been the topic of research in many phytochemical and pharmacological laboratories because they contain structurally complex and biologically fascinating steroidal alkaloids. This review, citing 153 references, summarises the chemistry, bioactivity and geographical diversity of steroidal alkaloids isolated from Veratrum and Fritillaria species, so as to illustrate the chemo-diversity and biological significance of these alkaloids, along with their geographical distribution where this is discernible.

Characterization and identification of steroidal alkaloids in Fritillaria species using liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry

Liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (LC/ESI-QTOF-MS/MS) was performed to study the fragmentation behaviors of steroidal alkaloids from Fritillaria species, the antitussive and expectorant herbs widely used in traditional Chinese medicine. We propose, herein, a strategy that combining key diagnostic fragment ions and the relative abundances and amounts of major fragment ions (the ions exceeding 10% in abundance) to distinguish different sub-classes of Fritillaria alkaloids (FAs). It was found that hydrogen rearrangement and induction effects result in ring cleavage of the basic skeletons occurred in the MS/MS process and produced characteristic fragment ions, which are useful for structural elucidation. This method was finally used to investigate the primary steroidal alkaloids in the extracts of eight major Fritillaria species. As a result, 41 steroidal alkaloids (29 cevanine type, 1 jervine type, 6 veratramine type and 5 secosolanidine type alkaloids) were selectively identified in these Fritillaria species. Twenty-six compounds were unambiguously identified by comparing with the reference compounds and 15 compounds were tentatively identified or deduced according to their MS/MS data. Logical fragmentation pathways for different types of FAs have been proposed and are useful for the identification of these types of steroidal alkaloids in natural products especially when there are no reference compounds available.

Characterizing distribution of steroidal alkaloids in Fritillaria spp. and related compound formulas by liquid chromatography–mass spectrometry combined with hierarchial cluster analysis

Bulbus Fritillariae (BF), referred to the bulbs of several Fritillaria species (Liliaceae), is a commonly used antitussive and expectorant herb in traditional Chinese medicine (TCM). Due to the complexity of BF botanical origin in the herbal markets, it is urgently needed to develop a reliable method for species identification. Previous studies based on morphological and histological as well as molecular biological techniques have respective limitations. For the purpose of finding a possible discriminant method among Fritillaria species, 27 steroidal alkaloids in 17 Fritillaria species and 12 BF-containing compound formulas were identified and characterized by a high-performance liquid chromatography with mass spectrometry (HPLC-MS) method. The estimated relative composition of steroidal alkaloids was used to carry out a chemotaxonomical study on Fritillaria species by means of hierarchial cluster analysis. In addition, the characteristic occurring patterns of the examined bases were compared in an effort to distinguish the botanical origin of BF-containing compound formulas. The results demonstrated that the qualitative and quantitative differences in steroidal alkaloids were useful not only for chemotaxonomy in some medicinal Fritillaria species but also for species identification in compound formulas. The described method has important implications in quality control of BF-containing TCM preparations, allowing for the prevention of BF confusion, and also revealing the possible presence of adulteration.

Turbulent-flow chromatography coupled on-line to fast high-performance liquid chromatography and mass spectrometry for simultaneous determination of verticine, verticinone and isoverticine in rat plasma

A method based on the on-line turbulent-flow chromatography and fast high-performance liquid chromatography/mass spectrometry (TFC-LC/MS) was developed for sensitive and high throughput pharmacokinetic study of traditional Chinese medicines (TCMs). In this method, an on-line extraction column (Waters Oasis HLB) and a fast HPLC column with sub-2 microm particle size (Agilent Zorbax StableBond-C(18), 4.6 mm x 50 mm, 1.8 microm) in a column-switching set-up were utilized. HLB is a reversed-phase extraction column with hydrophilic-lipophilic balanced copolymer (2.1 mm x 20 mm, 25 microm particle size), which will exhibit some turbulent-flow properties at a high-flow rate. The method combines the speed and robustness of turbulent-flow extraction and the sensitivity and separation efficiency of fast HPLC-MS to analyze multiple and trace constituents of TCMs in plasma matrix. This method was successfully applied for pharmacokinetic study of verticine, verticinone and isoverticine, the chemical markers of Fritillaria thunbergii, after oral administration of total steroidal alkaloids extract of F. thunbergii to rats. Each plasma sample was analyzed within 7 min. The method demonstrated good linearity (R>0.999) ranged from 0.505 to 96.0 ng/mL with satisfactory accuracy and precision, and the lower limit of quantifications of verticine, verticinone and isoverticine were estimated to be 0.120, 0.595 and 0.505 ng/mL, respectively. These results indicate that the proposed method is fast, sensitive, and feasible for pharmacokinetic study of TCMs.

Stilbene glucoside inhibits the glucuronidation of emodin in rats through the down-regulation of UDP-glucuronosyltransferases 1A8: application to a drug-drug interaction study in Radix Polygoni Multiflori.

ETHNOPHARMACOLOGICAL RELEVANCE The integrated effects of herbal medicines were the outcome of all of the inherent components. Currently, few studies have focused on the multicomponent interactions in an herbal medicine to elucidate its pharmacological and/or toxicological effects. In this study, an attempt was made to investigate the interaction between stilbene glucosides and the anthraquinones contained in Radix Polygoni Multiflori (RPM) and to explore the interactions mechanism from the perspective of UDP-glucuronosyltransferase (UGT) regulation. MATERIALS AND METHODS The extract of RPM was separated into a stilbene glucoside fraction and a emodin fraction. A rapid high-performance liquid chromatography-mass spectrometry method was developed and validated to disclose the influence of stilbene glucoside on the pharmacokinetics of emodin in rats. Drug and Statistics 2.0 was used for the estimation of the pharmacokinetic parameters. Gene expression analysis in liver and intestinal tissues was performed by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. RESULTS The analytical method appeared to be suitable for the analysis of emodin with desirable linearity, accuracy, precision and stability, and the total analysis time was less than 2 min on a short column. Glucuronide of emodin, which is the major metabolite of emodin, was determined after β-glucuronidase hydrolysis. As the in vivo pharmacokinetic studies had indicated, the AUC, Cmax and T1/2 of emodin were increased after the stilbene glucoside treatment, and the glucuronidation of emodin was significantly inhibited. The mRNA levels from UGT1A8 and UGT1A2 were decreased by stilbene glucoside treatment. In contrast, the expression of UGT1A1, UGT1A6 and UGT1A9 mRNA was increased in the liver following treatment. CONCLUSIONS The influence of stilbene glucoside on the pharmacokinetics of emodin may be attributed to the inhibition of UGT1A8 mRNA expression. Thus, it is important to extend this research to deepen our understanding of the pharmacological and/or toxicological effects of RPM.

Determination of emodin in L-02 cells and cell culture media with liquid chromatography-mass spectrometry: application to a cellular toxicokinetic study.

The emodin-involved hepatotoxicity has been gaining increasing attention. The purpose of the present study was to evaluate the cytotoxicity of emodin on cultured human liver cells (L-02) and predict the possible relation between its cytotoxicity and cellular toxicokinetics. Cell viability and cell damage were assessed by Cell Counting Kit-8 (CCK-8) assay and phase-contrast microscopy, respectively. Cytotoxicity tests demonstrated a concentration- and time-dependent toxic effect of emodin on L-02 cells. Furthermore, emodin at concentration of 30μM led to a significant apoptosis in a time-dependent manner supported by the morphological changes of drug-treated cells. In addition, to elucidate the toxicokinetic characteristics of emodin, a highly sensitive and selective liquid chromatography-mass spectrometry (LC-MS) method was employed and validated for detecting the dynamic alteration of emodin in cells and cell culture media. The proposed method appeared to be suitable for the analysis of emodin with desirable linearity (r(2)>0.99), and satisfying precision being less than 8.7%. The range of recoveries of this method was 90.2-101.9%. The preliminary cellular toxicokinetic study revealed a time-dependent intracellular accumulation of emodin, which was consistent with its in vitro toxic effects. These findings confirmed the cytotoxicity of emodin against L-02 cells and displayed the cytotoxic manner of emodin in terms of its cellular uptake and accumulation in L-02 cells.

Simultaneous qualification and quantification of baccharane glycosides in Impatientis Semen by HPLC-ESI-MSD and HPLC-ELSD.

This study presents a high performance liquid chromatography (HPLC) with electrospray ionization mass spectrometric detection (ESI-MSD) and evaporative light scattering detection (ELSD) method for the simultaneous qualification and quantification of eight major baccharane glycosides, namely hosenlosides A, B, C, F, G, K, L, and M in Impatientis Semen, a Chinese herbal medicine derived from the seeds of Impatiens balsamina L. In order to achieve optimum performance, several extraction parameters (including extraction solvent, extraction mode, extraction time) were optimized. The baccharane glycosides were separated on a Shim-pack CLC-ODS column with gradient elution of water and methanol. Temperature for the ELSD drift tube was set at 98°C and the nitrogen flow rate was 2.7l/min. The unambiguous identities of the analytes were realized by comparing retention times and mass data with those of reference compounds. The developed method was fully validated in terms of linearity, sensitivity, precision, repeatability, recovery as well as robustness, and subsequently applied to evaluate the quality of 14 batches of Impatientis Semen commercial samples from different collections.

A segmental monitoring strategy based on variable wavelength detection for quality control of three Polygonaceae herbs.

Radix Polygoni Multiflori, Rhizoma et Radix Polygoni Cuspidati, and Radix et Rhizoma Rhei are the most frequently used traditional Chinese medicines in the family Polygonaceae. The three herbal medicines (HMs) contain similar types of compounds. In Chinese Pharmacopoeia, five high-performance liquid chromatography (HPLC) coupled with ultraviolet (UV) detection methods have been employed for their quality control. The aim of this study was to develop a simple and conventional strategy, segmental monitoring based on variable wavelength detection (VWD), for simultaneous quantification of phenolic acids, flavonoids, stilbenes and anthraquinones in the three chemically analogous HMs. Compared with the commonly used HPLC-diode array detection (DAD), the proposed method afforded desirable performance on linearity, precision and sensitivity. Additionally, a quadrupole time-of-flight mass spectrometry (QTOF-MS) was applied to the structural confirmation of analytes from complex matrices. Based on the chemical profiles and contents of the analyses, 27 samples from three HMs were well classified using the principal component analysis. The results of this study demonstrated the potential and applicability of segmental monitoring strategy based on VWD for comprehensive quality control of HMs.

Puqienine E: An angiotensin converting enzyme inhibitory steroidal alkaloid from Fritillaria puqiensis

Eight steroidal alkaloids, puqienine A, puqienine B, puqienine C, puqienine D, puqienine E, puqietinone, puqiedine and peimisine were isolated from Fritillaria puqiensis G. D. Yu et. G. Y. Chen, and their anti-hypertensive effect were assessed in vitro based on the inhibition of the purified angiotensin converting enzyme (ACE) using high-performance liquid chromatography assay. The results showed that puqienine E, puqienine B and puqienine A exhibited better inhibitory activity against ACE than others, with inhibition ratios of 70.2+/-0.5%, 24.7+/-0.5% and 20.4+/-2.8%, respectively at the concentration of 200 microM. The 50% inhibiting concentration of puqienine E was determined to be 68 microM.