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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., Competing Interests: The authors declare no conflict of interest.

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 β 2 -adrenoceptor, which mediates the aforementioned ailments. In this work, a column containing immobilized β 2 -adrenoceptor was prepared using a diazonium salt reaction. The bioactive compound collected from the β 2 -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 β 2 -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) × 10 5 and (2.80 ± 0.01) × 10 4  M -1 , respectively. Molecular docking analysis of the interaction between chlorogenic acid and β 2 -adrenoceptor indicated that the binding mainly occurred on Ser 169 , Ser 173 , and Phe 287 of β 2 -adrenoceptor. These results paved the way to screen bioactive compounds of other traditional medicines by receptor chromatography., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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 β 2 -adrenoceptor (β 2 -AR), angiotensin II type 1 (AT 1 ) and angiotensin II type 2 (AT 2 ) 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.

Drug-protein interaction analysis is pregnant in designing new leads during drug discovery. We prepared the stationary phase containing immobilized β 2 -adrenoceptor (β 2 -AR) by linkage of the receptor on macroporous silica gel surface through N,N'-carbonyldiimidazole method. The stationary phase was applied in identifying antiasthmatic target of protopine guided by the prediction of site-directed molecular docking. Subsequent application of immobilized β 2 -AR in exploring the binding of protopine to the receptor was realized by frontal analysis and injection amount-dependent method. The association constants of protopine to β 2 -AR by the 2 methods were (1.00 ± 0.06) × 10 5 M -1 and (1.52 ± 0.14) × 10 4 M -1 . The numbers of binding sites were (1.23 ± 0.07) × 10 -7 M and (9.09 ± 0.06) × 10 -7 M, respectively. These results indicated that β 2 -AR is the specific target for therapeutic action of protopine in vivo. The target-drug binding occurred on Ser 169 in crystal structure of the receptor. Compared with frontal analysis, injection amount-dependent method is advantageous to drug saving, improvement of sampling efficiency, and performing speed. It has grave potential in high-throughput drug-receptor interaction analysis., (Copyright © 2017 John Wiley & Sons, Ltd.)

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 × 10(5)  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.)

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 × 10(6)  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.)

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.

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.

A receptor affinity chromatographic selection method was developed for screening the bioactive compounds binding to beta(2)-adrenoceptor (beta(2)-AR) in Coptidis rhizome. The bioactive compounds were analyzed by molecular recognition with a beta(2)-AR affinity column. The retention compounds eluted from the beta(2)-AR column were separated online with reverse-phase high-performance liquid chromatography by column switching technology, and identified by a coupled ion-trap mass spectrometer. Four compounds were screened as the bioactive compounds of Coptidis rhizome and identified as 2,9,10-trimethoxy-3-hydroxyl-protoberberine (jateorhizine), 2,3-methylenedioxy-9-methoxy-protoberberine, 2,3,9,10-tetramethoxy-protoberberine (palmatine) and 2,3-methylenedioxy-9,10-dimethoxy-protoberberine (berberine). The association constants of jatrorrhizine, palmatine and berberine to the beta(2)-AR were determined by the zonal elution method with standards. Berberine and palmatine had only one type of binding site on the immobilized beta(2)-AR. Their association constants were (2.28+/-0.11)x10(4)/M and (3.00+/-0.10)x10(4)/M, respectively. Jatrorrhizine had at least two type of binding sites on the immobilized beta(2)-AR, and the corresponding association constants were (2.20+/-0.09)x10(-4)/M and (6.78+/-0.001)x10(5)/M., (2010 Elsevier B.V. All rights reserved.)

A method is developed for the analysis of allantoin, gallic acid, dihydromelittoside, loganin, paeoniflorin, benzoylpaeoniflorin, and paeonol in Liuwei Dihuang tablets by high-performance liquid chromatography (HPLC)-UV-mass spectrometry (MS)-MS. Gradient elution with methanol-acetonitrile-water-formic acid solvent system is employed in the HPLC-electrospray ionization-MS study. The positive-ion ESI mode is suitable for these compounds. The peaks of gallic acid, loganin, dihydromelittoside, paeoniflorin, benzoylpaeoniflorin, and paeonol are identified by their mass spectra and the fragments of their MS-MS spectra. Allantoin, gallic acid, loganin, paeoniflorin, and paeonol are simultaneously determined by UV detection at 210 nm for quantitative purposes.