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The identification of bioactive compounds in complex matrices remains a major challenge due to the lack of highly efficient and specific methods. This work developed an approach based on high-performance affinity chromatography to identify the potential antitussive compounds from Zhisou oral liquid . The main methods include the synthesis of immobilized beta2-adrenoceptor by a one-step method, the screening and identification of the potential bioactive compounds by the receptor column coupled with mass spectrometry, and the binding mechanism analysis of the compounds to the receptor by the in vivo experiment, injection amount dependent method and molecular simulation. We identified the potential bioactive compounds of Zhisou oral liquid as glycyrrhizic acid, platycodin D, tuberostemonine, and hesperidin. In vivo experiment showed that the combinational utilization of the four compounds was possible to present an equivalent antitussive effect to the formula. The docking results demonstrated that hydrogen bonds and Van der Waals forces were the main forces to drive the binding of the four compounds to beta2-adrenoceptor. We concluded that the four compounds are the effective components in Zhisou oral liquid. The proposed strategy is possible to provide an alternative for the development of highly efficient methods to pursue the bioactive compounds of complex matrices.

Immobilized protein has advanced many areas like drug discovery. While this field evolved rapidly over the three decades, the immobilization platform for G-protein-coupled receptor (GPCR) remains unpromising due to its instability under the relatively harsh conditions of current methodologies. Taking beta2-adrenoceptor (β2-AR) as an example, we presented here a general strategy for immobilization of GPCRs by combing his-tag trap system, conformation-specific aptamer, and target binding induced DNA hybridization. Morphology characterization by diverse assays confirmed a monolayer of β2-AR on the microsphere surface. Radio-ligand binding assay and immuno-transmission electron microscopy displayed desirable ligand- and antibody-binding activities. Owing to the competitive strand displacement during the immobilization, the method proved to be capable of sensitively and directly determining the receptor density on the surface which enormously challenges most of the reported assays. A case study of chromatography using the immobilized receptor as stationary phase exhibited a demonstrable conformation specificity that enables the selective recognition of the receptor agonists or antagonists. This method is possible to turn into a general strategy for immobilization of GPCRs with defined orientation, conformation, function, and density, thus paving a way to precisely realize the receptor-ligand binding interaction and screen the receptor agonist or antagonist with high efficiency.

Protein immobilization is particularly significant in proteomics, interactomics, and in vitro drug screening. It is an essential primary step for numerous biological techniques that rely on immobilized proteins with controlled orientation, high conformational stability, and high activity (CHH). These have challenged the current immobilization strategy and demanded increasing efforts for an efficient method to meet the CHH immobilization in a single step. Herein, we proposed a covalent inhibitor-based, one-step method for G protein-coupled receptor (GPCR) immobilization inspired by the covalent reaction between an epidermal growth factor receptor (EGFR)-tag and its inhibitor ibrutinib. We immobilized endothelin receptor A (ETA) containing a fusion EGFR tag onto an ibrutinib-coated macroporous silica gel. The immobilized ETA proved to have demonstrable ligand-binding activity and specificity, thus resulting in a chromatographic technology allowing receptor-ligand interaction analysis and lead identification. Such immobilization method is attractable, owing to the properties of mild reacting conditions, fast rate, high yield, and good stability of the conjugated protein. It will be applicable to biochips, biosensors, and biocatalysts.

The past decade has witnessed the great promise of strategies for ligand discovery based on surface-immobilized GPCRs. We present here a method for preparation of immobilized GPCRs. Key features include covalent immobilization with high specificity and robust application in drug-receptor interaction analysis and ligand screening. In our example assay using beta2-adrenergic receptor (β2-AR), the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hAGT) fusion receptor expressed in Escherichia coli was directly captured onto polyethylene glycol polyacrylamide (PEGA) resin. We observed even distribution and physiological functions of β2-AR on the resin. The immobilized β2-AR as a stationary phase enabled us to rapidly determine the binding of four drugs to β2-AR. By coupling this assay to mass spectrometry, we screened rosmarinic acid as a bioactive compound targeting β2-AR in Fructus Perillae. We concluded that O6-benzylguanine derivative-functionalized supporter is promising for specific immobilization of hAGT-tagged proteins; immobilized receptor chromatography has great potential in screening receptor-binding leads from herbal plants or traditional medicine recipes.

The rapid growth of demands for drug discovery has necessitated the ongoing pursuit of new methods for specific ligands screening and identification. This work combined receptor-affinity chromatography (RAC) with high-throughput sequencing techniques to rapidly screen and identify the specific ligands. By this method, immobilized angiotensin II type I receptor (AT1R) and endothelin receptor A (ETAR) based on RAC were utilized for lead screening from a DNA-encoded library. The specific ligands of AT1R (ligand A1, A2) and ETAR (ligand B1, B2) were synthesized after decoding by high-throughput sequencing techniques. The dissociation rate constants (kd) of ligand A1, A2 to AT1R and B1, B2 to ETAR were 9.65 × 10−4, 31.1 × 10−4 and 0.66, 1.22 s−1 by peak profiling assay. The association constant (KA) to the receptors of four ligands was 5.4 × 106, 3.3 × 106 and 1.6 × 106, 2.2 × 105 by injection amount dependent method. The kinetic and thermodynamic parameters of the four specific ligands are similar to those of the positive drugs. This indicates that they are promising to drug candidates. The druggability of the four ligands through pharmacokinetic investigation by HPLC-MS/MS presented desired pharmacokinetic behavior including the fast absorption, the relatively slow elimination. These results, taking together, indicated that the RAC combined with high-throughput sequencing techniques can screen and identify the specific ligands according to various proteins, thus creating a general strategy for rapid discovery of promising drug candidates.

The kinetic study in drug-receptor interaction analysis is of great significance for the efficacy and safety of a drug. In previous work, we established a one-step method to immobilize the endothelin receptor A (ETA) on the surface of macroporous silica gel through a covalent interaction between the epidermal growth factor tyrosine kinase (EGFR) at the C terminal of ETA and the covalent inhibitor ibrutinib. The affinity stationary phase was used for lead screening and investigating the thermodynamic binding of ligands and ETA. Herein, we intend to introduce the ETA column in the kinetic study of the receptor and three specific ligands (bosentan, macitentan, and ambrisentan) as well as three leads (ferulic acid, berberine, and palmatine) from herbal medicines. Three classical methods in affinity chromatography, nonlinear chromatography, peak decay, and peak profiling, were performed to determine the dissociation rate constants of the ligands to ETA. A comparison of the three methods was made and the binding parameters were discussed. The three methods gave the same order of the dissociation constants between the six ligands and ETA. The results indicated that the immobilized ETA can be used for not only thermodynamic studies but also kinetic investigations, which provides a reliable and fast evaluation for drug-receptor interaction analysis.

The pursuit of drugs having improved therapeutic efficacy necessitates increasing research on new assays for screening bioactive compounds with multi-targets. This work synthesized a chromatographic stationary phase containing co-immobilized beta2-adrenergic receptor (β2-AR) and voltage dependent anion channel isoform 1 (VDAC-1) to achieve such purpose. Specific ligands of the two receptors (e.g. salbutamol, methoxyphenamine, ATP and NADH) were utilized to characterize the specificity and bioactivity of the column. Validated application of the stationary phase was performed by screening multi-target compounds of Rhodiola crenulata using high performance affinity chromatography coupled with ESI-Q-TOF-MS. By zonal elution, we identified salidroside as a bioactive compound simultaneously binding to β2-AR and VDAC-1. The compound exhibited the binding sites of 1.0 × 10−7 and 4.0 × 10−7 M on the β2-AR and VDAC-1. On these sites, the association constants were calculated to be 3.3 × 104 and 1.0 × 104 M−1. Molecular docking indicated that the binding of salidroside to the two receptors occurred on Ser169 and Phe255of β2-AR, and the channel wall of VDAC-1. Taking together, we concluded that the column containing co-immobilized receptors has potential for screening bioactive compounds with multi-targets from complex matrices including traditional Chinese medicines.

Responsive self-assembly is a general process in biological systems and is highly desired in engineered systems. DNA nanostructures provide a versatile molecular platform for studying such responsive self-assembly. Various triggers have been explored for DNA nanostructures. However, each trigger requires a unique mechanism for its response. This situation brings a great challenge to engineer the responsiveness. Herein, we propose an aptamer-based, allosteric mechanism for responsive DNA self-assembly. The aptamer-ligand binding causes the DNA motif to change its conformation and thus influences the motif assembly. With a model of an ATP aptamer, we have demonstrated the responsive assembly. Such responsive behavior, we believe, will be an important element for molecular machines, bioimaging/biosensing, and drug delivery.

Fine particulate matter is associated with adverse health effects, but exactly which characteristics of PM2.5 are responsible for this is still widely debated. We evaluated seasonal dynamics of the composition and chemical characteristics of PM2.5 in Zhuhai, China. PM2.5 characteristics at five selected sites within Zhuhai city were analyzed. Sampling began on January 10, 2015, and was conducted for 1 year. The ambient mass concentration, carbon content (organic and elemental carbon, OC and EC), level of inorganic ions, and major chemical composition of PM2.5 were also determined. Average concentrations of PM2.5 were lower than the National Ambient Air Quality Standard (NAAQS) 24-h average of 65 μg/m3. The daily PM2.5 concentration in Zhuhai city exhibited clear seasonal dynamics, with higher daily PM2.5 concentrations in autumn and winter than in spring and summer. Carbon species (OC and EC) and water-soluble ions were the primary components of the PM2.5 fraction of particles. Apart from OC and EC, chemical species in PM2.5 were mainly composed of NH4+ and SO42−. There was a marked difference between the summer and winter periods: the concentrations of OC and EC in winter were roughly 3.4 and 4.0 times than those in summer, while NH4+, SO42−, NO3−, and Na+ were 3.2, 4.5, 28.0, and 5.7 times higher in winter than those in summer, respectively. The results of chemical analysis were consistent with three sources dominating PM2.5: coal combustion, biomass burning, and vehicle exhaust; road dust and construction; and from reaction of HCl and HNO3 with NH3 to form NH4Cl and NH4NO3. However, additional work is needed to improve the mass balance and to obtain the source profiles necessary to use these data for source apportionment.

Although a comparatively robust method, immobilized protein-based techniques have displayed limited precision and inconsistent results due to a lack of strategy for the accurate selection of drug adsorption models on the protein surface. We generated the adsorption data of three drugs on immobilized beta-2-adrenoceptor (β2-AR) by frontal affinity chromatography–mass spectrometry (FAC-MS) and site-specific competitive FAC-MS. Using adsorption energy distribution (AED) calculations, we achieved the best adsorption models for the binding of salbutamol, terbutaline, and pseudoephedrine to immobilized β2-AR. The Langmuir model proved to be desirable for describing the adsorptions of salbutamol and terbutaline on immobilized β2-AR, while the bi-Langmuir model was favorable to characterize the adsorption of pseudoephedrine on the receptor. Relying on the accurate determination of association constants, we presented an efficient approach for β2-AR ligand screening based on the loss of breakthrough time of an indi...

Objective To understand the in vivo metabolic fate of 1,2,3,4,6-Penta-O-galloyl-β- d -glucose (PGG) naturally existed in many medicinal herbs and food plants such as Rhus chinensis, Paeonia suffruticosa, Paeonia lactiflora and Mango. Methods The metabolites of PGG in rat biofluids were characterized using high performance liquid chromatography combined with quadrupole time-of-flight tandem mass spectrometry (HPLC-QTOF-MS). Results Ten metabolites in urine, five metabolites in feces and two metabolites in plasma, were observed when the rats were administrated with a single intravenous injection of PGG (20 mg/kg). Conclusion PGG is firstly metabolized to gallic acid, then gallic acid undergoes sulfation, glucuronidation and methylation by rat liver. The determination of metabolites and the proposed metabolic pathway of PGG in vivo will be benefit to gain deeper insights into its pharmacological activities.

Drug discovery based on natural products like medicinal herbs remains challenging due to the technique limitations for rapidly screening and validating leads. To address the challenges, we employ the immobilized β2- adrenergic recepotor (β2-AR), an identified target of asthma, as the stationary phase in chromatographic column to screen compounds extracted from Stemonae Radix, Playtycodonis Radix, and Glycyrrhizae Radix et Rhizoma. To analyze binding properties of the extracted compounds to the immobilized receptors, we measured their retention behavior in the receptor chromatography and compared with six clinical asthma drugs. We identified tuberostemonine, platycodin D, and glycyrrhizic acid as the potential leads against asthma by our β2-AR chromatography coupled with mass spectrum (MS). The association constants of the three compounds to β2-AR were 2.85 × 10−5, 2.55 × 10−4, and 4.07 × 10−6 M with the dissociation rate constants of 6.91 ± 0.35, 11.88 ± 0.60, and 9.49 ± 0.64 min−1, respectively. Tuberostemonine, a pentacyclic Stemona alkaloids, presented the most optimum values of binding efficiency index (BEI) and surface efficiency index (SEI) as close to the diagonal of SEI–BEI optimization plane when it is compared with platycodin D, glycyrrhizic and the six clinical drugs. Our results suggest that tuberostemonine is a promising natural product to be developed for treating asthma because it exhibits better drug-like binding properties to β2-AR than the clinical drugs. As such, we demonstrate a chromatographic strategy to identify bioactive natural products based on the β2-AR immobilization, which can be widely adopted to screen natural products from mixture of herbal extracts.

The β2 adrenergic receptor (β2-AR) is a prototypical family A G protein-coupled receptor (GPCR) and an excellent model system for studying the mechanism of GPCR activation. Purified β2-AR was immobilized on macroporous silica gel to obtain liquid chromatographic stationary phase. The resulting phase was packed into a stainless steel column (4.6 × 50 mm, 7 μm) and used for on-line chromatographic system. When column oven temperature increased from 20.0 °C to 40.0 °C, uncomplete separate chromatographic peaks of ephedrine and pseudoephedrine as receptor conformational probe were gradually merged into one peak, meanwhile retention time and resolution of the probes were reduced correspondingly, which suggested that temperature could regulate protein conformation. Temperature-induced conformational change of immobilized β2-AR, especially changes at higher temperatures, indicated that constructed receptor chromatography could simulate fever disease state of human body and clarify receptor conformation change at pathological condition. At the same time this study could also provide new ideas for screening active components in pathological conditions.

The pursuit of new ligands binding to muscarinic-3 acetylcholine receptor (M3R) is viewed as challenging due to the lack of screening methods with high efficiency. To address such challenges, this work developed and characterized an approach to the rapid discovery of M3R ligands using the immobilized receptor as the chromatographic stationary phase. We fused haloalkane dehalogenase (Halo) as a tag at the C-terminus of M3R. The fusion M3R was immobilized on 6-chlorocaproic acid-activated ammino-microspheres by the specific covalent reaction between the Halo-tag and the linker. Comprehensive characterizations of the immobilized M3R were performed by scanning electron microscope, X-ray photoelectron spectroscopy, and the investigation on the binding of three specific ligands to the receptor. The feasibility of the immobilized M3R in complex matrices was tested by screening the bioactive compounds in Zhisou oral liquid, assessing the interaction between the screened compounds and the receptor using zonal elution, and evaluating the in vivo activity of the targeted compounds. The results evidenced that the immobilized M3R has high specificity, good stability, and the capacity to separate M3R ligands from complex matrices. These allowed us to identify naringin, hesperidin, liquiritigenin, platycodin D, and glycyrrhizic acid as the potential ligands of M3R. The association constants of the five compounds to M3R were 4.44 × 104, 1.11 × 104, 7.20 × 104, 4.15 × 104, and 3.36 × 104 M−1. The synergistic application of the five compounds exhibited an equivalent expectorant activity to the original formula. We reasoned that the current method is possible to provide a highly efficient strategy for the discovery of receptor ligands.

Owing to the promising clinical efficacy and relatively simple composition, Shuang-Huang-Lian prescription is widely prescribed for the treatment of acute upper respiratory tract infection and acute bronchitis in practice. This necessitates the understanding of the bioactive compounds of the prescription and their binding mechanism to β2 -adrenoceptor, which mediates the aforementioned ailments. In this work, a column containing immobilized β2 -adrenoceptor was prepared using a diazonium salt reaction. The bioactive compound collected from the β2 -adrenoceptor column was identified as chlorogenic acid by using high-performance liquid chromatography coupled with ion trap mass spectrometry. Using an injection amount dependent method, chlorogenic acid proved the binding to β2 -adrenoceptor through two kinds of sites. The numbers of the sites were (1.42 ± 0.03) × 10-8 and (9.06 ± 0.49) × 10-8 M. The association constants were (2.72 ± 0.01) × 105 and (2.80 ± 0.01) × 104 M-1 , respectively. Molecular docking analysis of the interaction between chlorogenic acid and β2 -adrenoceptor indicated that the binding mainly occurred on Ser169 , Ser173 , and Phe287 of β2 -adrenoceptor. These results paved the way to screen bioactive compounds of other traditional medicines by receptor chromatography.

Despite promising progress in series of techniques for drug discovery, the number of approved drugs has decreased in recent decades due to the lack of high efficient methods for candidate screening. In this work, immobilized beta2-adrenergic receptor was utilized to develop a new method for screening bioactive compounds from complex prescription, involving oriented immobilization of beta2-adrenergic receptor through diazonium reaction, the fast affinity extraction of bioactive compounds from Xie-Bai-San and the identification of these compounds by time of flight mass spectrometry. Valine, 4-hydroxyl resveratrol and kukoamine B were identified as the bioactive compounds specifically binding to beta2-adrenergic receptor in Xie-Bai-San. The binding of these compounds to the receptor occurred on four amino acid residues, viz., Asp 113, Ser 204, Ser 207, and Phe 290 in the crystal structure of beta2-adrenergic receptor. Hydrogen bond and hydrophobic force were believed to drive the binding of the compounds to beta2-adrenergic receptor on these residues. The results indicated that the proposed methodology has advantages on rapid, high throughput, and specific analysis, meanwhile, it has the capacity to probe the binding sites of the screened bioactive compounds. Fast affinity extraction methodology is expected to become a powerful alternative, which is superior in rapid screening of bioactive compounds from complex matrices such as traditional Chinese medicine.

We report a strategy for programmed DNA self-assembly that is favorable in terms of both thermodynamics and kinetics. In a previous study, it has been demonstrated that DNA self-assembly is primarily driven by thermodynamics and the assembly kinetics is not considered. To reach such stable states at equilibria, prolonged annealing duration is needed. In addition, there are cases where the desired structures could not compete with alternative structures. For example, a single-stranded DNA with a palindromic sequence quickly folds into a one-stranded hairpin instead of forming a two-stranded DNA duplex. Given that most of the DNA tiles are multi-stranded complexes, the kinetic trap represents a challenge to DNA self-assembly. To overcome this problem, we have developed a one-stranded motif that can intramolecularly and quickly fold from a single DNA strand and can be programmed to assemble into a range of nanostructures, including a one-dimensional (1D) ladder, a 1D chain, a two-dimensional (2D) array, and a three-dimensional (3D) triangular prism. All structures have been characterized by polyacrylamide gel electrophoresis (PAGE) and atomic force microscopy (AFM) imaging.

Objective To determine the distribution of metabolites in the root barks of different tree peony cultivars for quality assessment. Methods Seven tree peony phenotypic cultivars with different colors were systematically analyzed using NMR-based metabolomics. Results A total of 16 metabolites from their methanol extracts were simultaneously identified and quantified, including one primary metabolite (sucrose) and 15 secondary ones (acetophenones, phenolics, monoterpene glycosides, flavonoids, and unsaturated fatty acids). The quantitative data indicated that sucrose (90-180 mg/g) and acetophenones (15-100 mg/g), and non-phenolics, monoterpene glycosides, flavonoids, and unsaturated fatty acids (2-15 mg/g) were the major metabolites in these tree peony cultivars. The significantly increasing levels of paeonoside with bioactivity were observed in “Xiangyu”, “Wujinyaohui”, “Roufurong”, “Yaohuang”, “Zhaofen”, “Doulű”, and “Yingrihong” in order. Opposite trends in the levels of paeonoside and paeonol were observed in “Xiangyu” and “Yingrihong”, suggesting that the changes of the secondary metabolites in plants were influenced by primary metabolites, such as sucrose/glucose, and the different physiological processes occurred in different tree peony cultivars. Conclusion “Yingrihong” with red flower has the highest medicine quality whereas “Xiangyu” with white flower has the worst one based on the content of paeonoside.

As the first and key step of traditional Chinese medicine (TCM)-guided drug development, lead discovery necessitates continuous exploration of new methodology for screening bioactive compounds from TCM. This work intends to establish a strategy for rapidly recognizing β2-adrenergic receptor (β2-AR) target compounds from the fruit of Siraitia grosvenorii (LHG). The method involved immobilization of β2-AR onto amino-microsphere to synthesize the receptor column, the combination of the column to high-performance liquid chromatography (HPLC) to screen bioactive compounds of LHG, the identification of the compounds by HPLC coupled with mass spectrometry (MS), and the evaluation of druggability through pharmacokinetic examination by HPLC–MS/MS. Mogroside V was screened and identified as the β2-AR-targeted bioactive compounds in LHG. This compound exhibited desired pharmacokinetic behavior including the time to reach peak plasma concentrations of 45 min, the relatively low elimination of 138.5 min, and the high bioavailability. These parameters indicated that mogroside V has a good druggability for the development of new drugs fighting β2-AR-mediated respiratory ailments like asthma. The combination of the methods in this work is probably becoming a powerful strategy for screening and early evaluating the bioactive compounds specifically binding to G-protein-coupled receptor target from complex matrices including TCM.

In previous work, we have established a one-step method to immobilize halo-tagged proteins onto microspheres through the covalent bond formed between the halo-tag and the halide linkers on the support surface. We observe extremely tailed peaks of most of drugs on the immobilized proteins, which is reasoned by the nonspecific interaction between the linkers and the drugs. To prove this, the current work designed five different halide linkers for the immobilization of beta2-adrenoceptor (β2-AR). We applied the immobilized receptor to systematically realize the effects of these halide linkers on drug-receptor interaction by analyzing peak profiles of five drugs. The retention times and the half-widths of the drugs appeared to be negatively correlated to the atom numbers of the linkers in the range of 6–13 atoms. Subsequent increase of linker atoms resulted in reduced retention times and wider peaks of the drugs. Applying identical linker length, we observed clear reduced retention times and half-widths of the five drugs than the linker in the absence of oxygen atom. Such improvement was dominated by the number of oxygen atoms. These indicated that linker S-4 (2-(2-(2-(2-chloroethoxy) ethoxy) ethoxy) acetic acid) was optimal to eliminate the unwanted non-specific interactions. In comparison with the columns prepared by linker S-1 (6-chlorocaproic acid) and histidine tagged β2-AR, the drugs on the linker S-4 column gave greater dissociation rate constants (e.g. 60.3±0.3 s-1 for salbutamol), which is closer to the data in literatures. Taking together, we concluded that optimization of the linker structure plays particular role in reducing the non-specific interaction between the immobilized protein and the drugs, thereby making the determination of drug-protein interaction more reliable.

High-performance affinity chromatography is limited by its high cost and high pressure. Paper is made up of porous fiber networks and has the properties of low cost, ease of fabrication, and biodegradable. Due to these advantages, herein, we immobilized beta2-adrenoceptor (β2-AR) onto the surface of the polytetrafluoroethylene membrane, a paper-based material, and constructed a G protein-coupled receptor (GPCR)-in-paper chromatographic platform. This platform was characterized by Fourier transform infrared spectroscopy, fluorescence analysis, X-ray photoelectron spectroscopy, and chromatographic studies. These morphological and elemental analysis showed that β2-AR was successfully immobilized on the paper surface. The specific drugs have good retentions on the GPCR-in-paper chromatographic platform. The association constants of salbutamol, terbutaline and bambuterol to β2-AR were calculated to be 2.02 × 104 M−1, 1.15 × 104 M−1, 1.75 × 104 M−1 by adsorption energy distribution, which were in good line with the values from frontal analysis, zonal elution and previous literatures. We demonstrated that the GPCR-in-paper platform was cost-effective, easy to be modified for protein immobilization, and applicable in the receptor-drug interaction analysis. We believe such a platform sheds new light on paper chromatography for receptor-drug interaction analysis and other applications.

Microglia-mediated neuroinflammation plays a crucial role in the pathophysiological process of multiple neurological disorders such as ischemic stroke, yet lacks effective therapeutic agents. Previously, we discovered one novel synthetic compound, tanshinol borneol ester (DBZ), possesses anti-inflammatory and anti-atherosclerotic activities, whereas little is known about its effects in CNS. Therefore, the present study aims to explore the effects and potential mechanism of DBZ on neuroinflammation and microglial function. Our studies revealed that DBZ significantly inhibited NF-κB activity, suppressed the production of pro-inflammatory mediators meanwhile promoted M2 mediators expression in LPS-stimulated BV2 cells and mouse primary microglia cells. DBZ also exhibited antioxidant activity by enhancing Nrf2 nuclear accumulation and transcriptional activity, increasing HO-1 and NQO1 expression, and inhibiting LPS-induced ROS generation in BV2 cells. Importantly, the anti-neuroinflammatory and antioxidant effects of DBZ above were reversed by Nrf2 knockdown. Additionally, DBZ ameliorated sickness behaviors of neuroinflammatory mice induced by systemic LPS administration, and significantly reduced infract volume, improved sensorimotor and cognitive function in rats subjected to transient middle cerebral artery occlusion (tMCAO); besides, DBZ restored microglia morphological alterations and shifted the M1/M2 polarization in both murine models. Mechanistically, DBZ-induced Nrf2 nuclear accumulation and antioxidant enzymes expression were accompanied by increased level of p-Akt(Ser473) (activation) and p-GSK3β(Ser9) (inactivation), and decreased nuclear level of Fyn both in vitro and in vivo. Pharmacologically inhibiting PI3K or activating GSK3β markedly increased nuclear density of Fyn in microglia cells, which blocked the promoting effect of DBZ on Nrf2 nuclear accumulation and its antioxidant and anti-neuroinflammatory activities. Collectively, these results indicated the effects of DBZ on microglia-mediated neuroinflammation were strongly associated with the nuclear accumulation and stabilization of Nrf2 via the Akt(Ser473)/GSK3β(Ser9)/Fyn pathway. With anti-neuroinflammatory and antioxidant properties, DBZ could be a promising new drug candidate for prevention and/or treatment of cerebral ischemia and other neuroinflammatory disorders.

Immobilized protein makes a profound impact on the development of assays for drug discovery, diagnosis and in vivo biological interaction analysis. Traditional methods are enormously challenged by the G-protein coupled receptor ascribed to the loss of receptor functions. We introduced a β2-adrenergic receptor (β2-AR) aptamer into the immobilization of the receptor. This was achieved by mixing the receptor conjugated silica gel with cell lysates containing the receptor. We found that the aptamer-directed method makes immobilized β2-AR good stability in seven days and high specificity of ligand recognition at the subtype receptor level. Feasibility of the immobilized β2-AR in drug-receptor interaction analysis was evaluated by injection amount-dependent method, nonlinear chromatography, and peak decay analysis. Salbutamol, methoxyphenamine, ephedrine hydrochloride, clorprenaline, tulobuterol, bambuterol, propranolol and ICI 118551 bound to the receptor through one type of binding sites. The association constants presented good agreement within the three methods but exhibited clear differences from the data by radio-ligand binding assay. Regarding these results, we concluded that the aptamer-directed method will probably become an alternative for reversible and site-specific immobilization of GPCRs directly from complex matrices; the immobilized receptor is qualitative for drug-receptor interaction analysis.

Human and animal studies have shown that the colonic concentrations of lipid peroxidation products, such as 4-hydroxynonenal (4-HNE), are elevated in inflammatory bowel disease (IBD). However, the actions and mechanisms of these compounds on the development of IBD are unknown. Here, we show that a systemic treatment of low-dose 4-HNE exacerbates dextran sulfate sodium (DSS)-induced IBD in C57BL/6 mice, suggesting its pro-IBD actions in vivo. Treatment with 4-HNE suppressed colonic expressions of tight-junction protein occludin, impaired intestinal barrier function, enhanced translocation of lipopolysaccharide (LPS) and bacterial products from the gut into systemic circulation, leading to increased activation of Toll-like receptor 4 (TLR4) signaling in vivo. Furthermore, 4-HNE failed to promote DSS-induced IBD in Tlr4-/- mice, supporting that TLR4 signaling contributes to the pro-IBD effects of 4-HNE. Together, these results suggest that 4-HNE exacerbates the progression of IBD through activation of TLR4 signaling, and therefore could contribute to the pathogenesis of IBD.

Traditional Chinese medicine (TCM) represents a valuable resource for lead compounds discovery. Given the complexity of TCM components, analytical methods play a key role in novel drug development. In our study, we established a high specific and reliable bio-active components screen system, where β2 adrenergic receptor (β2-AR) was immobilized on silica by non-covalent bonds and packed into a stainless steel column (4.6 × 50 mm, 7 μm) to form β2-AR chromatography column. The column was further coupled with high performance liquid chromatography-time of flight tandem mass spectrometry (TOF-MS/MS). By utilizing this strategy, we successfully identified four β2-AR-targeting compounds: tetrahydroberberine, tetrahydrocolumbamine, fumarine and corydaline from Corydalis Rhizome. The association constants between β2-AR and tetrahydroberberine (9.04 × 104/M) as well as fumarine (4.30 × 104/M) were determined by frontal chromatography. We also found that these two compounds shared the identical binding site on immobilized β2-AR with corresponding concentrations of 6.67 × 10−4 M and 5.88 × 10−4 M, respectively. The newly established method represents an efficient tool to identify the target specific natural compounds.

Obesity is associated with enhanced colonic inflammation, which is a major risk factor for colorectal cancer. Considering the obesity epidemic in Western countries, it is important to identify novel therapeutic targets for obesity-induced colonic inflammation, to develop targeted strategies for prevention. Eicosanoids are endogenous lipid signaling molecules involved in regulating inflammation and immune responses. Using an LC-MS/MS–based lipidomics approach, we find that obesity-induced colonic inflammation is associated with increased expression of soluble epoxide hydrolase (sEH) and its eicosanoid metabolites, termed fatty acid diols, in colon tissue. Furthermore, we find that pharmacological inhibition or genetic ablation of sEH reduces colonic concentrations of fatty acid diols, attenuates obesity-induced colonic inflammation, and decreases obesity-induced activation of Wnt signaling in mice. Together, these results support that sEH could be a novel therapeutic target for obesity-induced colonic inflammation and associated diseases.

The bioactive ingredients in Semen sinapis were rapidly screened by immobilized β2-adrenoceptor (β2-AR) and target-directed molecular docking. The methods involved the attachment of β2-AR using any amino group in the receptor, the simultaneous separation and identification of the retention compounds by high-performance affinity chromatography; the binding mechanism of the interesting compound to the receptor was investigated by zonal elution and molecular docking. Sinapine in Semen sinapis was proved to be the bioactive compound that specifically binds to the immobilized receptor. The association constant of sinapine to β2-AR was determined to be 1.36 × 105 M−1 with a value of 1.27 × 10−6 M for the number of binding sites. Ionic bond was believed to be the driving force during the interaction between sinapine and β2-AR. It is possible to become a powerful alternative for rapid screening of bioactive compounds from a complex matrix such as traditional Chinese medicine and further investigation on the drug–receptor interaction. Copyright © 2015 John Wiley & Sons, Ltd.

Although green fluorescence protein (GFP) and its antibody are widely used to track a protein or a cell in life sciences, the binding behavior between them remains unclear. In this work, diazo coupling method that synthesized a new stationary GFP was oriented immobilized on the surface of macro-porous silica gel by a phase. The stationary phase was utilized to confirm the validation of injection amount-dependent analysis in exploring protein-protein interaction that use GFP antibody as a probe. GFP antibody was proved to have one type of binding site on immobilized GFP. The number of binding site and association constant were calculated to be (6.41 ± 0.76) × 10(-10) M and (1.39 ± 0.12) × 10(9) M(-1). Further analysis by molecular docking showed that the binding of GFP to its antibody is mainly driven by hydrogen bonds and salt bridges. These results indicated that injection amount-dependent analysis is capable of exploring the protein-protein interactions with the advantages of ligand and time saving. It is a valuable methodology for the ligands, which are expensive or difficult to obtain.

Drought stress during the reproductive stage is one of the most important constraints on growth and productivity of soybean. There is compelling evidence that physiological and molecular approaches can effectively improve drought resistance in soybean, but strategies derived from soil management are poorly documented, especially under field conditions. In this study, we combined data from field experiments and used multivariate regression analyses to investigate factors determining soybean yield variability over two growing seasons with drought stress during the reproductive stage. Phosphorus application and soil available phosphorus explained a significant proportion of the variation in soybean yield under drought stress. As a whole, soybean fields that received adequate P supply had greater yields than those that did not. However, excessive P application significantly decreased soybean yield. These results suggest that P control is critical to mitigate soybean yield losses resulting from drought at the reproductive stage.

Fluxes of carbon dioxide (CO2) and methane (CH4) from hydroelectric and water supply reservoirs are receiving increasing attention around the world with a number of research groups having undertaken measurements of these emissions across a range of lakes and reservoirs located in different climates and landscapes. The use of floating chambers (aka flux chambers) is the most common technique for direct measurement of these fluxes. However, the relative performance of different measurement systems, especially different chamber designs, is not well documented. We report the results of an international workshop held in June 2012 at Three Gorges Dam, China, to compare measurements performed by four groups with extensive chamber monitoring experience: the Chinese Academy of Science (China), CSIRO (Australia), SINTEF (Norway), Hydro-Quebec/Environnement Illimite (Canada). A fifth group, Eawag (Switzerland), performed hydroacoustic surveys to detect ebullition in the water column. We recommend CH4 as a more suitable trace gas for comparing methodologies due to its relative stability in the surface layer of the water column, for example, it is not subject to significant diurnal changes due to photosynthesis and respiration. Measured fluxes agreed to within 20% between the four teams suggesting that the shape and dimensions of the floating chambers and the chamber gas flow rates (i.e., chamber residence time) did not have an appreciable systematic effect on the measured fluxes for the relatively low wind speeds prevalent at the reservoir. The CO2 and CH4 fluxes measured during the workshop agree well with previous measurements in Three Gorges Reservoir.

This approach utilized N,N′-bis(carboxymethyl)-L-lysine (ANTA) coordinated to bivalent metal cation Ni2+, leaving free coordination sites for the reversible binding of gene recombinant histidine-tagged β2-adrenoceptor onto macropore silica. The amount of transient metal nickel ion on the support was determined by atomic absorption spectrophotometry. The novel protein oriented immobilization β2-AR column was evaluated by five β2-adrenoceptor agonists, applying frontal analysis. The association equilibrium constant for ligands on the column was 1.98 × 104 M−1 for salbutamol, 3.43 × 104 M−1 for clenbuterol, 2.09 × 104 M−1 for tulobuterol, 1.84 × 104 M−1 for terbutaline, 1.71 × 104 M−1 for methoxyphenamine and corresponding concentrations at binding sites were 7.46 × 10−6 M, 1.82 × 10−5 M, 2.16 × 10−5 M, 8.29 × 10−6 M and 3.88 × 10−5 M, respectively. The results obtained from breakthrough and nonlinear fitting indicated that all the drugs have a single binding site on the β2-adrenoceptor column. The present combined histidine-tagged protein method was reliable and exact in revealing interactions between receptor and drugs.

Although the association constant and the number of binding sites of prazosin to α1A-adrenoceptor were determined by high performance affinity chromatography (HPAC) in our previous work, the thermodynamic behaviors and the binding mechanism of the drug to immobilized α1A-adrenoceptor remained unclear. This work intended to address the issue by HPAC and molecular docking. The investigations involved the determination of association constants by frontal analysis at different temperatures, the calculation of enthalpy, entropy and free energy changes, the examination of mobile phase composition on the binding parameters and the site-directed molecular docking. The changes of enthalpy, entropy and free energy during the interaction were −20.79 kJ mol−1, −59.28 J mol−1 K−1 and −2.4 kJ mol−1, respectively. The binding of prazosin to α1A-adrenoceptor was an endothermic process with an increase in entropy. This reaction was mainly driven by hydrogen bonds. The ionic strength of the mobile phase provided a positive response to the values of association constants, while the power of hydrogen and the concentration of isopropyl in the mobile phase showed a negative trend. Ser203 and Ser192 in the fifth transmembrane segment of the receptor were the positions for the formation of hydrogen bonds. It is possible to utilize the immobilized receptor to determine the mechanism of drug–receptor interactions.

N-Phenylpiperazine derivatives are widely used as clinical drugs for fighting diseases related to the cardiovascular system by mediating the signal pathway of α1-adrenoceptor. The binding mechanism of nine N-phenylpiperazine derivatives to α1A-adrenoceptor was explored using molecular docking and high performance affinity chromatography. The methodology involved homology modelling of the three dimensional structure of α1A-adrenoceptor, predication of the binding behaviors using LIBDOCK and investigation of the thermodynamic behaviors of the binding by frontal analysis. Molecular docking results showed that Asp106, Gln177, Ser188, Ser192 and Phe193 of the receptor were the main binding sites for the nine N-phenylpiperazine derivatives binding to α1A-adrenoceptor. The binding was driven by formation of hydrogen bonds and electrostatic forces. The affinity of these derivatives for the receptor depended on the functional groups of an ionizable piperazine, hydrogen bond acceptor and hydrophobic moiety in the ligand structures. Frontal analysis indicated that the association constants of these compounds for the receptor were determined by their structural deviations in the above-mentioned functional groups. Thermodynamic studies presented negative enthalpy and Gibbs free energy changes with a positive entropy change, providing proof that the binding of the derivatives to α1A-adrenoceptor was mainly driven by electrostatic forces. This result was in line with the binding mechanism predicted by molecular docking. It is possible to explore the binding mechanism of drug candidates specifically binding to α1A-adrenoceptor using receptor chromatography.

Investigations of drug-protein interactions have advanced our knowledge of ways to design more rational drugs. In addition to extensive thermodynamic studies, ongoing works are needed to enhance the exploration of drug-protein binding kinetics. In this work, the beta2-adrenoceptor (β2-AR) was immobilized on N, N’-carbonyldiimidazole activated amino polystyrene microspheres to prepare an affinity column (4.6 mm × 5.0 cm, 8 μm). The β2-AR column was utilized to determine the binding kinetics of five drugs to the receptor. Introducing peak profiling method into this receptor chromatographic analysis, we determined the dissociation rate constants (kd) of salbutamol, terbutaline, methoxyphenamine, isoprenaline hydrochloride and ephedrine hydrochloride to β2-AR to be 15 (±1), 22 (±1), 3.3 (±0.2), 2.3 (±0.2) and 2.1 (±0.1) s−1, respectively. The employment of nonlinear chromatography (NLC) in this case exhibited the same rank order of kd values for the five drugs bound to β2-AR. We confirmed that both the peak profiling method and NLC were capable of routine measurement of receptor-drug binding kinetics. Compared with the peak profiling method, NLC was advantageous in the simultaneous assessment of the kinetic and apparent thermodynamic parameters. It will become a powerful method for high throughput drug-receptor interaction analysis.

An approach is established for the specific immobilization of GPCRs from cell lysates that circumvents labor intensive purification procedures and minimize loss of activity., Protein immobilization techniques play an important role in the development of assays for disease diagnosis and drug discovery. However, many of these approaches are not applicable to transmembrane proteins. G protein-coupled receptors (GPCRs) are the largest protein superfamily encoded by the human genome and are targeted by a quarter of all prescription drugs. GPCRs are highly dynamic and sensitive to changes in the ambient environment, and current immobilization methodologies are not suitable for GPCRs. We used haloalkane dehalogenase (Halo) as an immobilization tag fused to the β2-adrenoceptor (β2-AR), angiotensin II type 1 (AT1) and angiotensin II type 2 (AT2) receptors. The engineered Halo-tag covalently binds to a specific substrate chloroalkane through Asp 106 in the catalytic pocket. The Halo-tagged GPCRs were expressed in Escherichia coli at a suitable yield. Accordingly, we loaded cell lysate containing Halo-tagged GPCRs onto a macroporous silica gel coated with chloroalkane. Morphological characterization indicated a homogeneous monolayer of immobilized Halo-tagged GPCRs on the silica gel surface. The immobilized receptors proved to be surrounded by specific bound phospholipids including PG C18:1/C18:1. We observed a radio-ligand binding ability and ligand-induced conformational changes in the immobilized GPCRs, suggesting the preservation of bioactivity. This method is a one-step approach for the specific immobilization of GPCRs from cell lysates and validates that immobilized receptors retain canonical ligand binding capacity. Our immobilization strategy circumvents labor-intensive purification procedures and minimizes loss of activity. The immobilized receptors can be applied to high-throughput drug and interaction partner screening for GPCRs.

Introduction The distribution of metabolites in the different root parts of Cortex Moutan (the root bark of Paeonia suffruticosa Andrews) is not well understood, therefore, scientific evidence is not available for quality assessment of Cortex Moutan. Objective To reveal metabolomic variations in Cortex Moutan in order to gain deeper insights to enable quality control. Methods Metabolomic variations in the different root parts of Cortex Moutan were characterised using high-performance liquid chromatography combined with mass spectrometry (HPLC–MS) and multivariate data analysis. The discriminating metabolites in different root parts were evaluated by the one-way analysis of variance and a fold change parameter. Results The metabolite profiles of Cortex Moutan were largely dominated by five primary and 41 secondary metabolites . Higher levels of malic acid, gallic acid and mudanoside-B were mainly observed in the second lateral roots, whereas dihydroxyacetophenone, benzoyloxypaeoniflorin, suffruticoside-A, kaempferol dihexoside, mudanpioside E and mudanpioside J accumulated in the first lateral and axial roots. The highest contents of paeonol, galloyloxypaeoniflorin and procyanidin B were detected in the axial roots. Accordingly, metabolite compositions of Cortex Moutan were found to vary among different root parts. Conclusion The axial roots have higher quality than the lateral roots in Cortex Moutan due to the accumulation of bioactive secondary metabolites associated with plant physiology. These findings provided important scientific evidence for grading Cortex Moutan on the general market. Copyright © 2014 John Wiley & Sons, Ltd.

Surface-enhanced Raman scattering (SERS) spectra of P-Aminobenzoic Acid (PABA) adsorbed on the silver nano-particles were studied, respectively, in the silver colloidal solution and on the dried silver-coated filter paper. To further study the adsorption behaviors of PABA on the different substrates, we analyze the problem by means of theoretical calculation. Five models of PABA adsorbed on the surfaces of silver nano-particles were established. The Raman spectra of these five models using DFT-B3PW91 with lanl2dz were calculated. By comparing the theoretical values with the experimental values, we found that the calculated Raman frequencies of the models were in good agreement with experimental values, respectively. So we can believe that the simplified models are probably reasonable to describe some surface- enhanced Raman experiments.

The interaction between drugs and receptors is particularly important in revealing the drug acting mechanism and developing new leads. In this work, α 1-Adrenoceptor (α 1-AR) from HEK293 cell line is purified and immobilized on the surface of macro-pore silica gel to prepare an high-performance affinity chromatography stationary phase for the pursuit of drug–receptor interactions by competition zonal elution. Naftopidil is found to have only one type of binding site to α 1-AR with an association constant of 1.45 × 106 M−1 and a concentration of binding sites of 1.56 × 10−6 M, while terazosin hydrochloride proves to present two kinds of binding site on the receptor at which the association constants are determined to be 1.61 × 105 M−1 and 2.06 × 103 M−1, and the corresponding concentrations of the binding sites are 1.56 × 10−6 M and 1.11 × 10−3 M, respectively. It is concluded that the stationary phase containing attached α 1-AR can be used to realize the binding of a drug to the receptor.

A highly selective assay was developed for screening compounds that bind to the porcine recombinant β 2 -adrenoceptor (β 2 -AR) with affinity chromatography coupled to quadrupole time-of-flight mass spectrometry (Q-TOF–MS). The methodology involved selective screening with immobilized β 2 -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 β 2 -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 β 2 -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. β 2 -AR affinity chromatography was valuable in focusing attention on the further investigation of ferulic acid, HSYA, and naringin as β 2 -AR agonists.

Weak self-interaction plays an important role in interpreting the biomechanisms and modes of drug action. The structure-dependent self-association of five phenolic acids with various bioactivities, including danshensu (DSS), caffeic acid (CA), rosmarinic acid (RA), lithospermic acid (LA), and salvianolic acid B (SA), was investigated by 1H NMR. These phenolic acids have similar condensed structures, with a CA moiety and varying numbers of DSS moieties. The strengths of the self-association constants are in the order DSS

A new mathematical model and frontal analysis were used to characterize the binding behavior of caffeic acid to human serum albumin (HSA) based on high-performance affinity chromatography. The experiments were carried out by injecting various mole amounts of the drug onto an immobilized HSA column. They indicated that caffeic acid has only one type of binding site to HSA on which the association constant was 2.75 × 104/m. The number of the binding site involving the interaction between caffeic acid and HSA was 69 nm. The data obtained by the frontal analysis appeared to present the same results for both the association constant and the number of binding sites. This new model based on the relationship between the mole amounts of injection and capacity factors assists understanding of drug–protein interaction. The proposed model also has the advantages of ligand saving and rapid operation. Copyright © 2014 John Wiley & Sons, Ltd.

Affinity chromatography is the most widespread and widely accepted methodology for exploring drug-protein and protein-protein interactions. Despite the successful application of frontal analysis and zonal elution in affinity chromatography, research into the creation of new mathematical tools for data processing is encouraged due to these two methods' drawbacks of long analysis times and high ligand consumption. In this work, we created a novel mathematical model using the relationship between the molar amount of an injected solute and its capacity factor. We validated the method by analyzing the binding of drugs to human serum albumin (HSA) and β2-adrenoceptor (β2-AR). The association constants of omeprazole, propranolol and promethazine binding to HSA were determined to be (4.10±0.24)×10(4), (2.30±0.12)×10(4) and (1.24±0.14)×10(4)M(-1), respectively. These constants agreed with previously reported literature results of 4.60×10(4), 2.30×10(4) and 1.40×10(4)M(-1). Salbutamol, norepinephrine, isoprenaline, bamethane and methoxyphenamine were found to bind to β2-AR with association constants of (1.11±0.06)×10(3), (0.95±0.03)×10(3), (1.66±0.12)×10(3), (0.47±0.04)×10(3) and (0.43±0.02)×10(3)M(-1), respectively, which positively correlated to the negative logarithm of the dissociation constants obtained via radio-ligand binding assays. The proposed model is relatively fast and conserves ligand, and it has the potential to serve as an alternative method for rapidly revealing drug-protein and protein-protein interactions.

A method based on reaction with a diazonium salt was developed to immobilise oriented His-tagged protein onto silica gel. The binding efficiency of the phenylamine-group-coated gel was determined to be 65 %, providing a binding capacity of His-tagged protein up to the gram level. Using His-tagged β2-adrenoceptor (β2-AR) as a probe, we developed a new mathematical model to elucidate the interactions between the receptor and five ligands (methoxyphenamine, terbutaline, salbutamol, tulobuterol and fenoterol). These drugs proved to only have one type of binding site on the immobilised β2-AR, yielding higher association constants and numbers of binding sites than random attachment assays. The association constants determined by the new model positively correlated to the values from a radioligand binding method, with a regression equation of y = 1.75x - 7.18 and a correlation coefficient of 0.9807. The oriented method resulted in a high binding capacity and quantitative immobilisation of the His-tagged protein. The proposed model can be used to determine the interactions between the ligands and the immobilised protein with the advantages of drug and time saving.

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.

A pilot-scale process has been developed for green and scalable synthesis of (±)-β-(3,4-dihydroxyphenyl) lactic acid ((±)-DSS) and their two important derivatives, namely, (±)-IDHP [(±)-isopropyl 2-hydroxy-3-(3,4-dihydroxyphenyl)propanoate] and (±)-DBZ [(±)-bornyl 2-hydroxy-3-(3,4-dihydroxyphenyl)propanoate]. Subsequent hydrogenation has been carried out by employing Raney Ni as catalyst. The improved process results in higher yields of 47.5% for (±)-DBZ and 49.2% for (±)-IDHP compared to the initial process with a yield of 12% for (±)-DBZ and 18% for (±)-IDHP in our original medicinal chemistry route. Furthermore, kilograms of optical DBZ [(−)-S-DBZ and (+)-R-DBZ, >99% ee] and IDHP [(−)-S-IDHP and (+)-R-IDHP, >99% ee] have been produced by chiral high-performance liquid chromatography in good yield (>84%).

Self-competitive displacement is used to examine changes in the association constant and the binding site for berberine binding to immobilized β2-adrenoceptor (β2-AR) at different temperatures and using a varying mobile phase. The compound was confirmed to have a single kind of binding site to β2-AR under all the tested conditions. At 298.15 K, the association constant and the binding site are (2.12 ± 0.02) × 104 M−1 and (1.56 ± 0.02) × 102 M. Thermodynamic investigation indicates that the binding of berberine to β2-AR has a positive change in energy due to enthalpy and negative response to entropy changes. Accordingly, this interaction is thought to be an endothermal process with entropy increase. The binding of berberine to the receptor was found to be more sensitive to ionic strength than pH. The placing of 1-propanol up to 5.0% in the mobile phases generates an 18% decrease in retention for berberine. These results indicate that the immobilized receptor would probably be an alternative for exploring t...

The host–guest interaction between sulfobutylether-β-cyclodextrin (SBE-β-CD) and reserpine (RSP) is described using flow injection-chemiluminescence (FI-CL) and site-directed molecular docking methods. It was found that RSP could inhibit the CL intensity produced by a luminol/SBE-β-CD system. The decrease in CL intensity was logarithmic over an RSP concentration range of 0.03 to 700.0 nM, giving a regression equation of ∆I = 107.1lgCRES + 186.1 with a detection limit of 10 pM (3σ). The CL assay was successfully applied in the determination of RSP in injection, saliva and urine samples with recoveries in the range 93.5–106.1%. Using the proposed CL model, the binding constant (KCD-R) and the stoichiometric ratio of SBE-β-CD/RSP were calculated to be 7.4 × 106 M-1 and 1 : 1, respectively. Using molecular docking, it was confirmed that luminol binds to the small cavity of SBE-β-CD with a nonpolar interaction, while RSP targeted the larger cavity of SBE-β-CD and formed a 1 : 1 complex with hydrogen bonds. The proposed new CL method has the potential to become a powerful tool for revealing the host–guest interaction between CDs and drugs, as well as monitoring drugs with high sensitivity. Copyright © 2013 John Wiley & Sons, Ltd.

v/v) at a flow rate of 0.3 mL/min. A tandem mass spectrometric detection with an electrospray ionization (ESI) interface was conducted via collision induced dissociation (CID) under negative ionization mode. The MS/MS transitions monitored were m/z 343.0448 → m/z 313.2017 for usnic acid and m/z 153.1024 → m/z 136.2136 for protocatechuic acid (internal standard). The linear range was calculated to be 2.0-160.0 ng/mL with a detection limit of 3.0 pg/mL. The inter- and intra-day accuracy and precision were within ± 7.0%. Pharmacokinetic study showed that the apartment of usnic acid in vivo confirmed to be a two compartment open model. The method was fully valid and will probably be an alternative for pharmacokinetic study of usnic acid.