Junpeng Xing

Screening and Structural Characterization of α-Glucosidase Inhibitors from Hawthorn Leaf Flavonoids Extract by Ultrafiltration LC-DAD-MSn and SORI-CID FTICR MS

In vitro α-glucosidase inhibition assays and ultrafiltration liquid chromatography with photodiode array detection coupled to electrospray ionization tandem mass spectrometry (ultrafiltration LC-DAD-ESI-MSn) were combined to screen α-glucosidase inhibitors from hawthorn leaf flavonoids extract (HLFE). As a result, four compounds were identified as α-glucosidase inhibitors in the HLFE, and their structures were confirmed to be quercetin-3-O-rha- (1-4)-glc-rha and C-glycosylflavones (vitexin-2″-O-glucoside, vitexin-2″-O-rhamnoside and vitexin) by high-resolution sustained off resonance irradiation collision-induced dissociation (SORI-CID) data obtained by Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). Several other C-glycosylflavones (vitexin, isovitexin, orientin, isooriention) and their aglycones apigenin and luteolin were evaluated by in vitro assays, and were found to possess strong α-glucosidase inhibitory activities as well. Moreover, the substituent groups on the flavones had a great impact on the enzyme inhibition activity. C-3′-OH of the B-ring of flavones in particular increased the α-glucosidase inhibition activity, whereas C-glycosylations at C-6 or C-8 of the A ring weakened the inhibition activity.

Multi-residue method for fast determination of pesticide residues in plants used in traditional chinese medicine by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry.

An ultra-high-performance liquid chromatographic-tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous quantification and identification of 116 pesticide residues which were most widely used in plants used in Traditional Chinese Medicine (TCM) in 15 min has been developed and validated. Samples were extracted and cleaned up with modified QuEChERS method and detected by UHPLC-MS/MS under multiple reactions monitoring mode, and quantified by matrix-match calibration. The validation study was carried out on five different matrixes following DG SANCO/2007/3131 of the European Quality Control Guidelines. The linearity of the calibration was good between 5 and 100 ng ml⁻¹ concentration ranges, and the limits of quantification (LOQs) less than 0.01 mg/kg for most pesticides. The mean recoveries of almost all pesticides were in the range from 70% to 120% at three concentration levels ranging from 0.01 mg/kg to 0.1mg/kg with relative standard deviations (RSD) better than 15%. The method was applied on 138 real samples from 102 different kinds of Chinese herbal medicine. 95 positive samples were detected. This method is fast, robust, accurate, selective, sensitive and easy to operate.

Screening and Determination for Potential α‐Glucosidase Inhibitors from Leaves of Acanthopanax senticosus Harms by Using UF‐LC/MS and ESI‐MSn

INTRODUCTION Acanthopanax senticosus Harms is a typical Chinese herb with flavonoids existing in all parts of the plant but with the largest content in leaves. However, leaves have been neglected in past research. To investigate the potential use of leaves of A. senticosus Harms for discovering lead compounds to treat type 2 diabetes, the herb leaves were selected for screening the potential of α-glucosidase inhibitors. OBJECTIVE To screen for candidates of α-glucosidase inhibitors from leaves of A. senticosus Harms and evaluate the structure-activity relationship of the α-glucosidase inhibitors. METHODOLOGY Ultrafiltration liquid chromatography/mass spectrometry (UF-LC/MS) assay was developed for screening candidates of α-glucosidase inhibitors from leaves of A. senticosus Harms. The interesting compounds were identified by using reversed-phase high performance liquid chromatography with diode array detector and electrospray ionisation multiple-stage tandem mass spectrometry (RP-HPLC-DAD-ESI-MS(n) ), and confirmed by using electrospray ionisation Fourier transform ion cyclotron resonance multiple-stage tandem mass spectrometry (ESI-FT-ICR-MS(n)). Furthermore, the α-glucosidase inhibitory activity of the compounds detected was estimated using in vitro assays. RESULTS Eight compounds that might bind to α-glucosidase were screened and seven of them were identified successfully. The α-glucosidase inhibitory activity of the related compounds in leaves of A. senticosus Harms was determined. CONCLUSION The results obtained provided new information for the discovery of potential α-glucosidase inhibitors and the potential anti-diabetic application of the leaves of A. senticosus Harms.

Ultrahigh performance liquid chromatography–triple quadrupole mass spectrometry inhibitors fishing assay: A novel method for simultaneously screening of xanthine oxidase inhibitor and superoxide anion scavenger in a single analysis

Xanthine oxidase (XOD) inhibitors and superoxide anion scavengers play an important role in the treatment of gout and the inhibition of many diseases related to superoxide anion. The respective quantitation of uric acid and superoxide anion by traditional spectroscopic methods is routine in XOD inhibitors and superoxide anion scavengers screening at laboratories worldwide. In the present study, we established an ultrahigh performance liquid chromatography and triple quadrupole mass spectrometry (UHPLC-TQ-MS) method of higher accuracy and speed that combines screening of superoxide anion scavenger and XOD inhibitor in a single analysis by adding WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium salt) to the enzymatic reaction. We applied the established method to determine the XOD inhibitory activities and superoxide scavenging activities of some herbal extracts and compounds from natural products, which could be classified into six groups based on the results of the assay. Our innovative protocol is fast, accurate and robust. Moreover, it can eliminate false positive and false negative results which may occur in the traditional spectroscopic methods.

Probing the Interaction of Cisplatin with Cytochrome c by Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

As one of the most important platinum drugs, the interactions of cisplatin with proteins play very important roles in its anticancer action and side effects. Here, the interaction of cisplatin with cytochrome c was investigated using Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). On the basis of the high-resolution data, cytochrome c-Pt(NH(3))Cl was found to be the primary monoadduct. The platinated monoadducts were related to molar ratios of cytochrome c and cisplatin, and corresponding reaction pathways were proposed. Multiple binding sites of cisplatin on cytochrome c were directly determined by FTICR MS combined with trypsin digestion without liquid chromatography (LC) separation. Four binding sites (Met65, Met80, His18, and His33) for cisplatin on cytochrome c were identified. Moreover, hydrogen/deuterium exchange (H/DX) combined with FTICR MS provides the sensitive method to insight the small conformation change of cytochrome c induced by cisplatin. This strategy can be applied to comprehensive investigation of metallodrug/protein complexes.

Characterization of compounds and potential neuraminidase inhibitors from the n-butanol extract of Compound Indigowoad Root Granule using ultrafiltration and liquid chromatography-tandem mass spectrometry

The liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were used to identify the pharmacologically active n-butanol extract from Compound Indigowoad Root Granule. As a result, eighteen compounds belonging to various structural classes such as nucleosides, purines, flavonoids and amino acid were unambiguously identified. Then an in vitro neuraminidase (NA) inhibition assay was carried out to examine the inhibitory activity of the standard samples and extracts on NA. After which, ultrafiltration liquid chromatography with photodiode array detection coupled to electrospray ionization tandem mass spectrometry (ultrafiltration LC-MS/MS) was used to study NA inhibitory activity of standard flavones and n-butanol extract of Compound Indigowoad Root Granule. This method is highly selective and sensitive, and it could be used for characterization of bioactive compounds and botanical extracts. The result provides some enlightenment for the explanation of the antiviral activity of Compound Indigowoad Root Granule and some guidance for natural anti-influenza medicine development.

Determination of pesticide residues in ginseng by dispersive liquid-liquid microextraction and ultra high performance liquid chromatography-tandem mass spectrometry.

A procedure involving acetonitrile-based extraction combined with dispersive liquid-liquid microextraction (DLLME) and detection by ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was used for determination of 39 pesticides in ginseng. The extraction of pesticide residues in ginseng was performed with acetonitrile, applying QuEChERS methodology, and the extract was further disposed by DLLME method before analyzed by UHPLC-MS/MS. The average recoveries ranged from 70 to 120% for 82% of the analytes with RSD lower than 15%. The calibration curves obtained with blank matrices were linear with a correlation coefficient of over 0.99. The limits of detection were between 0.01 and 1.0μg/kg. Matrix effects were studied by comparing solvent calibration curves and matrix-matched calibration curves. The results indicate the feasibility of this method for the determination of 39 pesticides in ginseng.

Application of online microdialysis coupled with liquid chromatography-tandem mass spectrometry method in assessing neuroprotective effect of Rhizoma coptidis on diabetic rats

Monitoring the in vivo dynamics of neurochemicals is very important for studies on learning and memory impairment. However, drawbacks in conventional sampling and off-line analysis methods require the employment of a more satisfying system. We present an online microdialysis coupled with liquid chromatography-tandem mass spectrometry method (LC-MS/MS) to determine eight neurochemicals simultaneously, including glutamate, aspartic acid, γ-aminobutyric acid, serine, taurine and acetylcholine, as well as dopamine and serotonin. A self-assembled automated injector with a high-pressure-resisting six-port valve was utilized to control the switch of the two states between loading and injection, which potentially simplified the sample preparation procedures and avoided the restrictions offered by small sample volume. Good linearity (R2 > 0.997) was observed in each analyte dynamic range. The inter-day and intra-day precisions (RSD) were <15%, and the accuracy (RE) was from −13.84% to 11.16%. The overall matrix effect was within an acceptable range, from 87.79% to 111.71%. Morris water maze (MWM) test was performed to evaluate the differences of cognitive abilities among normal control, diabetes mellitus (DM) model and Rhizoma coptidis (R. coptidis)-treated DM group rats. Moreover, the developed method was applied for the further exploration of the R. coptidis efficacies on the eight analytes changes in the hippocampus of diabetic rats with mild cognitive impairment. It was found that an improvement in neurochemicals imbalance was observed in diabetic rats after treatment with R. coptidis. In conclusion, the developed online analysis method is selective, sensitive and accurate, which is suitable for in vivo neurochemicals monitoring and could be extended to pharmacology studies.

A strategy for identification and structural characterization of compounds from Gardenia jasminoides by integrating macroporous resin column chromatography and liquid chromatography-tandem mass spectrometry combined with ion-mobility spectrometry

In this paper, an analysis strategy integrating macroporous resin (AB-8) column chromatography and high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) combined with ion mobility spectrometry (IMS) was proposed and applied for identification and structural characterization of compounds from the fruits of Gardenia jasminoides. The extracts of G. jasminoides were separated by AB-8 resin column chromatography combined with reversed phase liquid chromatography (C18 column) and detected by electrospray ionization tandem mass spectrometry. Additionally, ion mobility spectrometry (IMS) was employed as a supplementary separation technique to discover previously undetected isomers from the fruits of G. jasminoides. A total of 71 compounds, including iridoids, flavonoids, triterpenes, monoterpenoids, carotenoids and phenolic acids were identified by the characteristic high resolution mass spectrometry and the ESI-MS/MS fragmentations. In conclusion, the IMS-MS technique achieved the separation of isomers in crocin-3 and crocin-4 according to their acquired mobility drift times differing from classical analysis by mass spectrometry. The proposed strategy can be used as a highly sensitive and efficient procedure for identification and separation isomeric components in extracts of herbal medicines.

SCREENING FOR α-GLUCOSIDASE INHIBITORS FROM COPTIDIS-REHMANNIAE HERB COUPLE BY USING ULTRAFILTRATION LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY

α-Glucosidase inhibitors have been used for the management of type 2 diabetes mellitus (T2DM) for a long time. The natural α-glucosidase inhibitors from Chinese herbal medicine have become an attractive therapeutic approach for treating T2DM due to their low toxicity. The assays for the discovery of compounds in natural product extracts, however, mostly needed labor-intensive and time-consuming purification. In the current study, the screening of α-glucosidase inhibitors from Coptidis-Rehmanniae herb couple which was composed of Rhizoma Coptidis and Radix Rehmanniae, was investigated by ultrafiltration liquid chromatography/mass spectrometry (UF-LC/MS). As a result, four compounds binding to α-glucosidase were observed and identified to be jatrorrhizine, coptisine, palmatine, and berberine. Subsequently, the sustained off-resonance irradiation collision-induced dissociation Fourier transform mass spectrometry was performed for validation. In addition, the specific binding of ligands to α-glucosidase was demonstrated using UF-LC/MS and the ligands were ranked in order of affinity for α-glucosidase. The relative affinity order for the ligands was coptisine > jatrorrhizine > palmatine ≈ berberine, which indicated that coptisine and jatrorrhizine exhibited more potent α-glucosidase inhibitory activity compared to palmatine and berberine. Therefore, UF-LC/MS might provide not only a powerful tool for screening α-glucosidase inhibitors in complex samples but also a useful platform for bioactive compounds discovery of the efficient anti-diabetic drugs.