Hon-Yeung Cheung

Enhanced analysis of triterpenes, flavonoids and phenolic compounds in Prunella vulgaris L. by capillary zone electrophoresis with the addition of running buffer modifiers

A cyclodextrin-modified capillary zone electrophoresis method was developed for the separation and determination of three isomeric compounds (ursolic acid, oleanolic acid and betulinic acid), caffeic acid, p-coumaric acid, rosmarinic acid, rutin and quercetin. Without the addition of beta-cyclodextrin (beta-CD) and methanol, the separation of these analytes was poorly resolved. These eight compounds, however, were well separated from each other within 20 min with a borax running buffer (40 mM of borax, pH 9.4) containing 2mM beta-CD and 4% (v/v) methanol at the voltage of 25 kV, temperature of 25 degrees C and detection wavelength of 210 nm. The relative standard deviations (RSDs) of migration time ranged from 0.16 to 0.74% while those of the peak area ratios ranged from 2.17 to 4.61% for six determinations of the analytes at concentration of 10 and 25 microg mL(-1). The correlation coefficients of the calibration curves of the analytes were all >0.998, and the recoveries were from 96.8 to 103.6%. The method was successfully applied to determine these bioactive components in the samples of Prunella vulgaris L. and its beverage drink products. Our results reveal that only the isomeric compounds and rosmarinic acid could be detected in the spikes of P. vulgaris L.; other components were either too low to be detected or not present while only rosmarinic acid was detected in the beverage products.

Determination of bioactive diterpenoids from Andrographis paniculata by micellar electrokinetic chromatography

The present paper describes the development of a micellar electrokinetic chromatographic (MEKC) method for simultaneous determination of andrographolide, deoxyandrographolide and neoandrographolide in ethanol extracts of Andrographis paniculata. Separations were carried out in a fused-silica capillary tube with UV detection at 214 nm. Good separation was achieved using a 20 mM borate buffer, containing 20 mM sodium dodecyl sulphate and 10 mM sodium cholate, adjusted to pH 8.3 at an operating voltage of 25 kV, temperature of 35 degrees C and a hydrodynamic injection of 5 s. The method was validated with good correlation coefficients obtained (0.9986-0.9989) while relative standard deviation (RSD) of migration time was between 1.14 and 2.42. It is concluded that this method could be used for speedy and accurate qualitative and quantitative analysis of bioactive diterpenoids in andrographis herb and its derived products.

Rapid and simultaneous analysis of some bioactive components in Eucommia ulmoides by capillary electrophoresis

A micellar electrokinetic chromatography method was established for the qualitative and quantitative determination of three groups of bioactive components, iridoids, flavonoids and phenolic compounds, in Eucommia ulmoides. Of the eleven bioactive components being studied, ten were successfully separated in 50 mM boric acid buffer at pH 9.5, with 50 mM sodium dodecylsulfate and 4% 1-butanol, at a voltage of 20 kV, temperature of 20 degrees C and injection under high pressure at 138 kPa for 5 s in a fused-silica capillary with peak detection at 214 nm. A high reproducibility and good linearity was obtained. The relative standard deviations of the migration times in eight injections of the standards ranged from 0.64 to 1.88% and those of the corrected peak area ranged from 2.79 to 6.62%. A good linearity, with correlation coefficients in the range of 0.995-1.000, was obtained in the calibration curves of each standard from 1 to 50 ppm. The amount of these bioactive components in the bark and leaves of Eucommia ulmoides were determined.

A FRET-based Ratiometric Fluorescent Probe for Nitroxyl Detection in Living Cells

HNO has recently been found to possess distinct biological functions from NO. Studying the biological functions of HNO calls for the development of sensitive and selective fluorescent probes. Herein, we designed and synthesized a FRET-based ratiometric probe to detect HNO in living cells. Our studies revealed that the probe is capable of detecting HNO in a rapid and ratiometric manner under physiological conditions. In bioimaging studies, the probe displayed a clear color change from blue to green when treated with HNO.

Graphene based pipette tip solid phase extraction of marine toxins in shellfish muscle followed by UPLC–MS/MS analysis

Graphene is a novel carbonic material with great potentials for the use as sorbent due to its ultrahigh surface area. Herein, we report the use of graphene as sorbent in solid-phase extraction (SPE) using pipette tip as cartridge namely GPT-SPE, together with ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), for the analysis of lipophilic marine toxins (LMTs), including yessotoxins (YTX), okadaic acid (OA), dinophysistoxin-1 (DTX1), gymnodimine (GYM), spirolides-1 (SPX1), pectenotoxin-2 (PTX2) and azaspiracid-1 (AZA1) in shellfish. The GPT-SPE procedure was optimized and the performance of graphene was fully validated. Results with high-sensitivity and good reproducibility was obtained and compared with that of other sorbents like C18 silica, multi-walled carbon nanotubes (MWCNTs), commercial Oasis HLB, and Strata-X for the extraction of LMTs, which showed superiority and advantages of graphene, such as good recoveries, stability and compatibility with various solvents. In order to exhibit the potentials of graphene as an excellent sorbent material, 67 mussel samples from six coastal cities of China were analyzed. OA was found to be the dominant contaminant, while YTX was also detected with low level.

Computational study on the molecular inclusion of andrographolide by cyclodextrin.

Due to the poor water solubility of andrographolide (andro), an inclusion technique has been developed to modify its physical and chemical properties so as to improve its bioavailability. In contrast with the immense experimental studies on the inclusion complexes of andro:cyclodextrin, no computational study has so far been carried out on this system. In this work, preliminary docking experiments with AutoDock were performed. Density Functional Theory (DFT) and Austin Model 1 (AM1) calculations upon the docking instances were applied to investigate the two possible modes of molecular inclusions between andro and x-cyclodextrin (xCD, where x is α, β or γ). Atoms-in-Molecules (AIM) analysis based on the B3LYP/cc-pVDZ wavefunction was applied to verify the existence of the intermolecular hydrogen bonds. It was found that the most stable complex among the six possible inclusion complexes was the one formed between andro and βCD with andro’s decalin ring moiety wrapped by CD at a ratio of 1:1. The hydrogen bonds between andro and CD were responsible for the stability of the inclusion complexes. The calculated data were found to be consistent with the experimental results. Thus, the results of this study can aid new drug design processes.

Real-time monitoring of Bacillus subtilis endospore components by attenuated total reflection Fourier-transform infrared spectroscopy during germination

Chemical changes of particular Bacillus subtilis spore components were monitored by attenuated total reflection Fourier-transform infrared spectroscopy (ATR/FTIR) during spore germination on a ZnSe internal reflection element. Within minutes of the initiation of spore germination, significant changes in the amount of calcium dipicolinate (DPA-Ca) and proteins were noted in the wild-type strain. The changes in a germination mutant (strain 1G9, gerD) were similar to those in the wild-type strain, but the rates of change were slower. The changes in another germination mutant (strain 1G7, gerA) were very different from those in the first two strains: germination was slow and incomplete, and proteins and DPA-Ca remained unaltered throughout the course of the germination study. This technique thus offers a sensitive and non-destructive method for real-time monitoring of various cellular components during spore germination.

A chromatographic method for rapid and simultaneous analysis of codeine phosphate, ephedrine HCl and chlorpheniramine maleate in cough-cold syrup formulation.

The present paper describes a simple, accurate and precise reversed phase HPLC method for rapid and simultaneous quantification of codeine phosphate, ephedrine HCl and chlorpheniramine maleate in a cough-cold syrup formulation. Separations were carried out on a Zorbax XDB C8 column (150 x 4.6 mm ID), 5 microm particle size. A gradient elution system was developed using varying percentages of two mobile phases: methanol-glacial acetic acid-triethylamine (980:15:6 v/v) and water-glacial acetic acid-triethylamine (980:15:6 v/v). The elution of the analytes was achieved in less than 7 min with a flow rate of 1.5 ml/min. Detection was by UV absorbance at a wavelength of 254 nm. Quantification of the components in actual syrup formulations was calculated against the responses of freshly prepared external standard solutions. The method was validated and met all analysis requirements of quality assurance and quality control recommended by FDA of the USA.

Differential Actions of Chlorhexidine on the Cell Wall of Bacillus subtilis and Escherichia coli

Chlorhexidine is a chlorinated phenolic disinfectant used commonly in mouthwash for its action against bacteria. However, a comparative study of the action of chlorhexidine on the cell morphology of Gram-positive and Gram-negative bacteria is lacking. In this study, the actions of chlorhexidine on the cell morphology were identified with the aids of electron microscopy. After exposure to chlorhexidine, numerous spots of indentation on the cell wall were found in both Bacillus subtilis and Escherichia coli. The number of indentation spots increased with time of incubation and increasing chlorhexidine concentration. Interestingly, the dented spots found in B. subtilis appeared mainly at the hemispherical caps of the cells, while in E. coli the dented spots were found all over the cells. After being exposed to chlorhexidine for a prolonged period, leakage of cellular contents and subsequent ghost cells were observed, especially from B subtilis. By using 2-D gel/MS-MS analysis, five proteins related to purine nucleoside interconversion and metabolism were preferentially induced in the cell wall of E. coli, while three proteins related to stress response and four others in amino acid biosynthesis were up-regulated in the cell wall materials of B. subtilis. The localized morphological damages together with the biochemical and protein analysis of the chlorhexidine-treated cells suggest that chlorhexidine may act on the differentially distributed lipids in the cell membranes/wall of B. subtilis and E. coli.

Identification and quantification of base and nucleoside markers in extracts of Ganoderma lucidum, Ganoderma japonicum and Ganoderma capsules by micellar electrokinetic chromatography

The present paper describes the development of a micellar electrokinetic chromatographic method for the determination of nucleoside (adenosine, uridine) and base (uracil) markers in aqueous extracts of Ganoderma medicinal preparations. The markers were successfully separated within 10 min using an 80 mM borate buffer, with 25 mM sodium dodecyl sulfate adjusted to pH 9.0, an operating voltage of 22 kV, temperature of 20 degrees C and a hydrodynamic injection time of 5 s. Separations were carried out in a fused-silica capillary with peak detection by direct UV at 254 nm. Following semi-validation of the method, with each analyte showing a good linear relationship over a 0.2 to 20 ppm concentration range (correlation coefficients from 0.9986 to 0.9998), the amounts of the three markers in the various forms of Ganoderma were easily determined using a relatively simple extraction procedure.