Shixiang Wang

Research on antioxidant effects and estrogenic effect of formononetin from Trifolium pratense (red clover).

Antioxidant and estrogenic effects of formononetin on ovariectomized mice have been investigated in the present study. The adult female Kunming mice were divided into 5 groups: sham-operated group, ovariectomized group, stilbestrol replacement therapy group (0.20 mg/kg day), low-dose formononetin group (0.05 g/kg day) and high-dose formononetin group (0.5 g/kg day). The mice in the latter 4 groups were ovariectomized. The drug was given by oral administration for 6 months. Estrogenic effect was determined by the change of uterine weight, and oxidant effects were determined by the content of SOD, GSH-Px, CAT and MDA. The intake of formononetin increased the uterine weight of the mice significantly as well as the content of SOD, GSH-Px, CAT, and reduced MDA in body. Formononetin had obvious antioxidant effects and estrogenic effect, and the estrogenic effect was not dosage-related.

Thermodynamic study of the interaction between terbutaline and salbutamol with an immobilized β2-adrenoceptor by high-performance liquid chromatography

Investigation of the interaction between drugs and receptors is very important in revealing the biologic basis and mechanism of the drug, and designing new bioactive compounds. The beta(2)-adrenoceptor (beta(2)-AR) was purified from rabbit lung tissues and immobilized on the surface of macro-pore silica gel through covalent bonds to prepare the stationary phase. Binding isotherms of terbutaline and salbutamol were determined by frontal analysis and the perturbation method, respectively. On the basis of the model of binding isotherm assumed, zonal elution was used to investigate the binding interaction of the receptor with terbutaline and salbutamol. The two drugs had one type of common binding site on immobilized beta(2)-AR. Salbutamol had at least one other major binding region. The association constant for terbutaline was (9.76+/-0.67)x10(4)/M, and the concentration of the binding sites was (9.37+/-1.32)x10(-6)M. Under identical conditions, association constants for salbutamol at the two types of binding site were (1.11+/-0.08)x10(4)/M and (1.34+/-0.13)x10(3)/M, and the concentration of the binding sites was (5.46+/-0.35)x10(-6)M. Entropy increase was the main driving force for terbutaline and salbutamol to bind with beta(2)-AR. The associating reaction of terbutaline and beta(2)-AR was an exothermal process primarily from electrostatic interactions; hydrophobic force was the major factor contributing to the process for salbutamol.

Effects of ionic liquid and nanogold particles on high-performance liquid chromatography-electrochemical detection and their application in highly efficient separation and sensitive analysis of five phenolic acids in Xuebijing injection

A novel high performance liquid chromatography-electrochemical detector (HPLC-ECD) analytical system was developed in this study by integratedly utilizing ionic liquid (IL) of 1-butyl-3-methylimidazolium bromide and an additive of gold nanoparticles. The resulted pilot study was first performed to assess the effects of 1-butyl-3-methylimidazolium bromide and gold nanoparticles on the chromatographic characteristics of five phenolic acids in Xuebijing injection, including danshensu (DSS), protocatechuic acid (PA), protocatechuic aldehyde (PAH), hydroxy safflower yellow A (HSYA) and ferulic acid (FA). It was notable to observe that retainability of the phenolic acids were markly lowered by IL addition. Compared with the cases without IL addition, the retention times of DSS, PA, PAH, HSYA and FA have decreased 2.851, 1.532, 1.53, 0.818 and 0.552 min, respectively when 0.6% IL in the mobile phase. In addition, the corresponding theoretical plate numbers and peak areas for these compounds were significantly increased. Area response for DSS, PA, PAH, HSYA and FA were enhanced by 772%, 628%, 584%, 703% and 600%, respectively. It was observed that nano-gold catalysis power enabled peak areas of DSS, PAH, FA and PA to enhance 5.7, 6.2, 8.5 and 66.5 times relative to the case with addition of IL. Altogether, the optimized HPLC-ECD system was successfully applied to the pharmacokinetics study of Xuebijing injection with underlying applicability to in vivo and in vitro analysis of a variety of natural product from Chinese medicine plants, TCM formulae and associated patent TCM preparation.

Reversing P-glycoprotein-mediated multidrug resistance in vitro by α-asarone and β-asarone, bioactive cis–trans isomers from Acorus tatarinowii

P-Glycoprotein (P-gp), an ATP-binding cassette transporter, plays an important role in multidrug resistance (MDR). α-Asarone and β-asarone, bioactive cis–trans isomers found in Acorus tatarinowii Schott, were tested for their potential ability to modulate the expression and function of P-gp in Caco-2 cells. MTT assays revealed that both α-asarone and β-asarone significantly enhanced the vincristine-induced cytotoxicity to cells. β-Asarone was the most potent. Flow cytometry showed that α- and β-asarone increased Rhodamine 123 (Rh123) uptake and inhibited Rh123 efflux in Caco-2 cells in a concentration-dependent manner. Furthermore, P-gp expression and P-gp mRNA in cells were decreased by exposure to α- and β-asarone. In addition, β-asarone increased the inhibition of P-gp activity in cells more than α-asarone. Thus, α- and β-asarone effectively reversed MDR by inhibiting P-gp function and expression.

Effects of Borneol on Pharmacokinetics and Tissue Distribution of Notoginsenoside R1 and Ginsenosides Rg1 and Re in Panax notoginseng in Rabbits

The purpose of this study is to investigate the effects of Borneol on the pharmacokinetics of notoginsenoside R1 (NGR1) and the ginsenosides Rg1 (GRg1) and Re (GRe) in Panax notoginseng. Reversed phase high-performance liquid chromatography coupled with electrospray ion trap mass spectrometry was employed to determine the concentrations of the three compounds in rabbit plasma. In comparison with rabbits administrated Panax notoginseng extract alone, animals simultaneously taking Panax notoginseng extract and Borneol exhibited significant differences in pharmacokinetic parameters of NGR1, GRg1, and GRe, such as increasing their bioavailability. Quantities of NGR1, GRg1, and GRe in rabbit tissues were also increased after combining administration of Borneol. In addition, the apparent permeability coefficients (P app) of NGR1, GRg1, and GRe were raised by Borneol significantly in Caco-2 cells. However, no significant changes were observed in the efflux ratio (Er) of NGR1, GRg1 and GRe. These data indicate that Borneol has the properties of enhancing the intestinal absorption, increasing the distribution, and inhibiting the metabolism of NGR1, GRg1, and GRe. The underlying mechanism might be attributed to the loosening of the intercellular tight junction.

Simultaneous Determination of Volatile Constituents from Acorus tatarinowii Schott in Rat Plasma by Gas Chromatography-Mass Spectrometry with Selective Ion Monitoring and Application in Pharmacokinetic Study

A sensitive and specific gas chromatographic-mass spectrometry with selected ion monitoring (GC-MS/SIM) method has been developed for simultaneous identification and quantification of α-asarone, β-asarone, and methyl eugenol of Acorus tatarinowii Schott in rat plasma. Chromatographic separation was performed on a Restek Rxi-5MS capillary column (30 m × 0.32 mm × 0.25 μm), using 1-naphthol as internal standard (IS). MS detection of these compounds and IS was performed at m/z 178, 208, 208, and 144. Intra- and interday precisions of all compounds of interest were less than 10%. The recoveries are situated in the range of 92.4–105.2%. Pharmacokinetics of methyl eugenol confirmed to be one-compartment open model, α-asarone and β-asarone was two-compartment open model, respectively. The method will probably be an alternative to simultaneous determination and pharmacokinetic study of volatile ingredients in Acorus tatarinowii Schott.

Study of captopril pharmacokinetics in rabbit blood with microdialysis based on online generated Au nanoclusters and pepsin–captopril interaction in luminol chemiluminescence

A luminol–HAuCl4–pepsin (Pep) flow injection-chemiluminescence (FI-CL) system was explored to determine captopril (CAP) based on the CL intensity inhibition effect and applied to study CAP pharmacokinetics in rabbits with microdialysis. HAuCl4 and pepsin (Pep) could significantly enhance the luminol chemiluminescence (CL) intensity. It was found that sub-nanometre Au nanoclusters (AuNCs) were generated in the luminol–HAuCl4–Pep reaction solution. A possible mechanism for AuNCs generation is given. By means of the FI-CL and molecular docking (MD) methods, the Pep–CAP interaction was systematically studied. The results showed that CAP might enter into Pep active site Asp32 with the binding constant (K) 1.7 × 106 L mol−1, which could effectively inhibit the CL intensity. The CL intensity could be remarkably inhibited by CAP and the decrement of CL intensity was linearly correlated to the logarithm of CAP concentration in the range of 3.0 pmol L−1 to 0.1 μmol L−1, with a detection limit of 1.0 pmol L−1 (3σ). This proposed approach was successfully applied to determine CAP in rabbit’s blood during the 16 h after intragastric administration with an elimination ratio of 45.9% and recovery ratios from 89.0% to 112.0%. The pharmacokinetic results showed that CAP could be rapidly absorbed into blood with a peak concentration (Cmax) of 9.63 ± 1.45 μg mL−1 at a maximum peak time (Tmax) of 0.75 ± 0.08 h; the elimination half-life of 3.19 ± 0.13 h and the elimination rate constant of 7.27 ± 0.41 L g−1 h−1 in rabbits were derived, respectively.

Metabolomics study on the synergistic interaction between Salvia miltiorrhiza and Lignum dalbergiae odoriferae used as ‘Jun-Shi’ herbs in a S. miltiorrhiza recipe

A metabolomic method was established to investigate the plasma metabolic difference between healthy rabbits and rabbits with Qi-stagnancy and blood stasis. All rabbits were administrated with an extraction of Salvia miltiorrhiza and S. miltiorrhiza coupled with Lignum dalbergiae odoriferae. The main compounds in plasma samples were detected by high-performance liquid chromatography/diode array detector/electrospray-mass spectrometry (HPLC/DAD/ESI-MS). The data were analyzed by principal component analysis (PCA). The results showed that Qi-stagnancy and blood stasis had a close relationship with dopamine. In addition, the results also indicated that the major plasma metabolic difference between rabbits administrated with S. miltiorrhiza and S. miltiorrhiza coupled with Lignum dalbergiae odoriferae were 4-methylbenzamide, Danshensu and β-(3,4-dihydroxybenzene)-α-hydroxyl propanoic acid isopropyl ester. The above results could provide experimental evidence for the theory of traditional Chinese medicine.

Highly selective screening of the bioactive compounds in Huoxue capsule using immobilized β2-adrenoceptor affinity chromatography

A highly selective assay was developed for screening compounds that bind to the porcine recombinant β2-adrenoceptor (β2-AR) with affinity chromatography coupled to quadrupole time-of-flight mass spectrometry (Q-TOF-MS). The methodology involved selective screening with immobilized β2-AR, a highly accurate identification via Q-TOF-MS, and a functional evaluation of the screened compounds with a sensitive myograph system. Ferulic acid, hydroxysafflor yellow A (HSYA), and naringin were confirmed to be the bioactive compounds in Huoxue capsule that specifically bound to the β2-AR. These compounds produced a concentration-dependent relaxation of arteries that were contracted by treatment with phenylephrine, and the relaxation caused by these compounds was attenuated in the presence of ICI 118551, a type of β2-AR antagonist. Our data indicate that the use of an immobilized receptor is potentially an alternative method for the rapid screening of bioactive compounds in a complex matrix because of its high specificity. β2-AR affinity chromatography was valuable in focusing attention on the further investigation of ferulic acid, HSYA, and naringin as β2-AR agonists.

Liquid Chromatography/Quadrupole Time-of-Flight Mass Spectrometry for Identification of In Vitro and In Vivo Metabolites of Bornyl Gallate in Rats

Bornyl gallate (BG) is a potential drug candidate synthesized by the reaction of two natural products, gallic acid and borneol. Previous studies have strongly suggested that BG is worthy of further investigation due to antioxidant, antiatherosclerosis activities, and obvious activity of stimulating intersegmental vessel growth in zebrafish. This work was designed to elucidate the metabolic profile of BG through analyzing its metabolites in vitro and in vivo by a chromatographic separation coupled with a mass spectrometry. The metabolites of BG were characterized from the rat liver microsome incubation solution, as well as rat urine and plasma after oral administration. Chromatographic separation was performed on an Agilent TC-C18 column (250 mm × 4.6 mm, 5 μm) with gradient elution using methanol and water containing 0.2% (V : V) formic acid as the mobile phase. Metabolites identification involved analyzing the retention behaviors, changes of molecular weights and MS/MS fragment patterns of BG and the metabolites. Five compounds were identified as isomers of hydroxylated BG metabolites in vitro. The major metabolites of BG in rat urine and plasma proved to be BG-O-glucuronide and O-methyl BG-O-glucuronide. The proposed method confirmed to be a reliable and sensitive alternative for characterizing metabolic pathways of BG.