Hailin Wang

Fractionation and analysis of Artemisia capillaris Thunb. by affinity chromatography with human serum albumin as stationary phase

A method for the screening and analysis of biologically active compounds in traditional Chinese medicine is proposed. Affinity chromatography using a human serum albumin (HSA) stationary phase was applied to separate and analyze the bioactive compounds from Artemisia capillaris Thunb. Five major peaks and several minor peaks were resolved based on their affinity to HSA, two of them were identified as scoparone (SCO, 6,7-dimethoxycoumarin) and capillarisin (CAP). CAP shows a much higher affinity to HSA than SCO. The effects of acetonitrile concentration, eluent pH, phosphate concentration and temperature on the retention behaviors of several major active components were also investigated, and it was found that hydrophobicity and eluent pH play major roles in changing retention values. The results demonstrate that the affinity chromatography with a HSA stationary phase is an effective way for analyzing and screening biologically active compounds in traditional Chinese medicine.

Determination of ginsenoside Rg3 in plasma by solid-phase extraction and high-performance liquid chromatography for pharmacokinetic study.

A method using high-performance liquid chromatography (HPLC) and solid-phase extraction (SPE) is described for the determination of ginsenoside Rg3 in human plasma. A 2.5-ml volume of plasma was mixed with 2.5 ml 60% methanol aqueous solution, and centrifuged at 1100 g for 10 min, the supernatant fluid was further purified by SPE with 200 mg/5 ml 40 microns octadecyl silica and separation was obtained using a reversed-phase column under isocratic conditions with ultraviolet absorbance detection. The intra- and inter-day precision, determined as relative standard deviations, were less than 5.0%, and method recovery was more than 97%. The lower limit of quantitation, based on standards with acceptable RSDs, was 2.5 ng/ml. No endogenous compounds were found to interfere with analyte. A good linear relationship with a regression coefficient of 0.9999 in the range of 2.5 to 200 ng/ml was observed. This method has been demonstrated to be suitable for pharmacokinetic studies in humans. Method development for determination of drug with low UV absorption by SPE and HPLC is also discussed.

Synthesis of a silica-bonded bovine serum albumin s-triazine chiral stationary phase for high-performance liquid chromatographic resolution of enantiomers

A novel method of synthesizing protein chiral stationary phase (protein-CSP) is proposed with 2,4,6-trichloro-1,3,5-triazine as the activator. The bovine serum albumin (BSA) based chiral columns (150 x 4.6 mm I.D.) were prepared successfully within 8 h. With tryptophan as the probe solute, it was observed that the BSA immobilized by this method had a better ability to distinguish enantiomers than that activated by glutaric dialdehyde. This may be due to the well-maintained BSA conformation and the larger amount of BSA immobilized on the silica gel. The BSA-CSP prepared by this method was relatively stable under experimental conditions, and the resolution of 13 chiral compounds was achieved. The coupling reaction in this method is mild, reliable and reproducible; it is also suitable for the immobilization of various biopolymers in the preparation of bioreactor, biosensor and affinity chromatography columns.

On-line characterization of the activity and reaction kinetics of immobilized enzyme by high-performance frontal analysis

A microreactor by immobilized trypsin on the activated glycidyl methacrylate-modified cellulose membrane packed column was constructed. Immobilized trypsin mirrored the properties of the free enzyme and showed high stability. A novel method to characterize the activity and reaction kinetics of the immobilized enzyme has been developed based on the frontal analysis of enzymatic reaction products, which was performed by the on-line monitoring of the absorption at 410 nm of p-nitroaniline from the hydrolysis of N-alpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA). The hydrolytic activity of the immobilized enzyme was 55.6% of free trypsin. The apparent Michaelis-Menten kinetics constant (Km) and Vmax values measured by the frontal analysis method were, respectively, 0.12 mM and 0.079 mM min(-1) mg enzyme(-1). The former is very close to that observed by the static and off-line detection methods, but the latter is about 15% higher than that of the static method. Inhibition of the immobilized trypsin by addition of benzamidine into substrate solution has been studied by the frontal analysis method. The apparent Michaelis-Menten constant of BAPNA (Km), the inhibition constant of benzamidine (Ki) and Vmax were determined. It was indicated that the interaction of BAPNA and benzamidine with trypsin is competitive, the Km value was affected but the Vmax was unaffected by the benzamidine concentration.

Binding of sulfamethoxazole to human serum albumin studied by a combined technique of microdialysis with liquid chromatography

Abstract A new, simple and fast method for the determination of the interaction parameters of sulfamethoxazole (SMZ) to human serum albumin (HSA) has been developed by utilizing a microdialysis sampling technique combined with liquid chromatography (LC). The drug and protein were mixed in different molar ratios in 0.067 M potassium phosphate buffer, pH 7.4, and incubated at 37 °C in a water-bath. Then the microdialysis probe was put in the SMZ-HSA solution and sampled at a perfusion rate of 1 μl/min. The concentration of SMZ in the microdialysates was determined by reversed-phase liquid chromatography. The recovery ( R ) was also determined in vitro under similar conditions, R is about 41.8% with an RSD of about 2.3%. The association constant ( K ) and the number of the binding sites on one HSA molecule ( n ) are calculated by three equations, the values of nK estimated by three methods are quite similar. The values found for K and n are 3.24 × 10 3 M −1 and 3.04, respectively.

Screening and Analysis of Biologically Active Compounds in Angelica sinensis by Molecular Biochromatography

SummaryA novel strategy for the screening and analysis of biologically active compounds in traditional Chinese medicine by molecular biochromatography is proposed. Molecular biochromatography with human serum albumin (HSA) immobilized on silica as stationary phase was used to screen and analyse the bioactive compounds in the typical Chinese medicine ofAngelica sinensis (Oliv.) Diels. Ten peaks showed retention on this column, which is based on their affinity for HSA. Ferulic acid and liguistilide were identified as the principal active components, which agrees very well with the results in the literature. A quality control method was also developed based on the simultaneous determination the concentrations of ferulic acid and liguistilide in solutions ofAngelica sinensis (Oliv.) Diels extracted with water and methanol. It was observed that the concentrations of ferulic acid and liguistilide in solution extracted with methanol were 2 and 53 times higher, respectively, than those with water. It was shown that molecular biochromatography is an effective way of analysing and screening biologically active compounds in traditional Chinese medicine.

Chemical and Toxicological Characterization of Halobenzoquinones, an Emerging Class of Disinfection Byproducts

Halobenzoquinones (HBQs), a new class of disinfection byproducts (DBPs), occur widely in treated drinking water and recreational water. The main concern regarding human exposure to DBPs stems from epidemiological studies that have consistently linked the consumption of chlorinated drinking water with an increased risk of developing bladder cancer. The U.S. Environmental Protection Agency and Health Canada have set regulations on the amount of DBPs in drinking water to minimize the risk. However, these regulated DBPs do not account for the increased risk of bladder cancer because they have different target organs or lower magnitudes of risk based on animal carcinogenesis studies. Because of the pervasive exposure to DBPs, identification of DBPs relevant to human health has become one of the important research targets to address DBP-associated health concerns. Quantitative structure-toxicity relationship (QSTR) analysis has predicted HBQs to be potential bladder carcinogens. Therefore, this perspective focuses on the chemical and toxicological characterization of HBQs. In vitro cytotoxicity experiments have shown that HBQs induce greater cytotoxicity and/or greater developmental toxicity than most of the regulated DBPs. Cellular mechanistic studies indicate that HBQs are capable of producing reactive oxygen species (ROS) either within cells or in solution, depleting cellular glutathione levels, and influencing cellular antioxidant enzymes, which further induces oxidative stress and oxidative damage to cellular proteins and DNA. Oxidative damage to DNA was demonstrated in the form of significant increases in cellular levels of 8-hydroxydeoxyguanosine (8-OHdG), DNA strand breaks, and apurinic/apyrimidinic (AP) sites. HBQs can also form DNA adducts, affect genome-wide DNA methylation, and inhibit DNA repair enzymes. These findings demonstrate that HBQs are highly cytotoxic and potentially genotoxic and carcinogenic, although in vivo data corroborating this is not available. To fully understand the potential adverse health effects and cancer risk due to HBQ exposure, multidisciplinary research is required regarding human exposure, health risk assessment, and toxicological mechanisms of HBQs.

Analysis of terpene compounds in Cimicifuga foetida L. by reversed-phase high-performance liquid chromatography with evaporative light scattering detection.

An RP-HPLC method with evaporative light scattering detection (ELSD) was developed for the analysis of terpene compounds in traditional Chinese medicine. Actein, 27-deoxyactein and cimicifugoside in a typical Chinese medicine of Cimicifuga foetida L. were quantitatively analyzed. Comparing ELSD with UV detection under the same eluent conditions, the former showed better sensitivity and a more stable baseline. The ELSD responses versus sample size of three terpenes and those in double logarithmic were investigated. The good calibration curves in double logarithmic coordinator for actein, 27-deoxyactein and cimicifugoside were obtained. Three solutions for the extraction of the terpene compounds were also compared, the results indicated that methanol-water (80:20) is the best among them. The method was applied to quantify actein, 27-deoxyactein and cimicifugoside in Cimicifuga foetida L. from Hunan, China. It was shown that ELSD is an effective detection method for the analysis of the non-volatile terpenes in traditional Chinese medicine.

Binding of metal ions with protein studied by a combined technique of microdialysis with liquid chromatography

A method has been developed for the determination of interactions of metal ions and protein by using microdialysis sampling technique combined with pre-column derivation and reversed-phase ion-pair liquid chromatographic (HPLC analysis. Cu(II), Zn(II) and human serum albumin (HSA) were chosen as model metal ions and protein, respectively. The mixed solutions of metal ions and HSA with different molar ratios buffered with 0.1 M Tris-HCl containing 0.1 M NaCl at pH 7.43 were sampled with a mirodialysis probe by keeping perfusion rate at 1 mul/min and the temperature at 37 degreesC. The free concentrations of metal ions in microdialysates were assayed by precolumn derivatization with meso-tetra(4-sulfophenyl)-porphyrin (TPPS4) followed ion-pair HPLC analysis. The recovery (R) of microdialysis sampling was measured in vitro under similar conditions as 65.74% for Cu(II), 70.45% for Zn(II) with R.S.D. below 3.2%. The primary binding constants and number of binding site estimated by the Scatchard plot analysis are 5.04 x 10(6) M-1 and 0.85 for Cu(II), and 9.87 x 10(6) M-1 and 1.10 for Zn(II), respectively. The competition of Cu(II) and Zn(II) at the second binding site on HSA was investigated, and it was observed that there is a second site on HSA to bind Cu(II) and Zn(II), the affinity of Cu(II) is stronger than that of Zn(II) to this second site of HSA

Determination of drug-protein interactions by combined microdialysis and high-performance liquid chromatography

SummaryA simple and rapid method for determination of the parameters of the interaction between drugs and protein, including the association constant and the number of binding sites, has been developed by use of a microdialysis sampling technique combined with high-performance liquid chromatography. The drug and protein (carbamazepine (5H-dibenz[b,f]flazepine-5-carboxamide, CBZ) and human serum albumin (HSA) were used as examples) were mixed in different molar ratios in 0.067 M potassium phosphate buffer, pH 7.4, and incubated at 37°C in a water-bath. The microdialysis probe was the used to sample the mixed CBZ-HSA solution at a perfusion rate of 1 μL min−1. The concentration of CBZ in the microdialysate was determined by reversed-phase high-performance liquid chromatography. Relative recovery (R), determined in vitro under similar conditions, was approximately 42.7%; theRSD ofR was approximately 1.85%. The estimated association constant (K) and the number of the binding sites,n, on one molecule of HSA were 1.06×104 M−1 and 0.880, respectively, which is in good agreement with the literature values determined by high-performance frontal analysis. The potential use of microdialysis is also discussed.