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

doi:10.1016/j.ab.2014.04.013

Analytical Biochemistry

Volume 457, 15 July 2014, Pages 1-7

https://doi.org/10.1016/j.ab.2014.04.013 Get rights and content

Abstract

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.

Section snippets

Materials and reagents

Huoxue capsule was acquired from the Xi’an Datang Pharmaceutical Group (Xi’an, China). Bambuterol, clorprenaline, phenylephrine, acetylcholine, Triton X-100, and ICI 118551 were purchased from Sigma (St. Louis, MO, USA). Naringin, hydroxysafflor yellow A (HSYA), and ferulic acid (purity > 98%) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products of China (lot nos. 110722-200610, 110722-200610, and 0773-9910, respectively). HPLC-grade solvents and

Purification of the β₂-AR

The purity of the obtained β₂-AR was 95.3%, as determined by analysis with sodium dodecyl sulfate polyacrylamide gel electrophoresis and high-performance size exclusion chromatography.

Selectivity and system suitability of β₂-AR column

The results of the positive control experiment revealed that the t_R values of bambuterol and clorprenaline that bound to the β₂-AR on the stationary phase were 5.51 and 6.86 min, respectively (Figs. 2B). However, the t_R values of prazosin and terazosin, which target the α-AR and were used as negative control drugs,

Conclusion

In this study, a method using porcine recombinant β₂-AR affinity chromatography on-line with LC/MS was established to screen for β₂-AR ligands from complicated systems such as natural plant extracts and traditional Chinese medicines. Using this method, ferulic acid, HSYA, and naringin were identified. This method can simultaneously recognize, separate, and identify multiple bioactive compounds and will be useful in drug discovery efforts using natural medicinal herbs to identify potential β₂-AR

Acknowledgments

This study was supported by grants from the National Natural Science Foundation of China (Major International [Regional] Joint Research Project, 81120108002; General Program, 81071765), the Specialized Research Fund for the Doctoral Program of Higher Education (20130201130008), the Program for Changjiang Scholars and Innovative Research Team in the University of China (IRT1174), the Program for Innovative Research Team of Shaanxi Province (2013KCT-24), and the Scientific Research Plan Projects

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

Abstract

Section snippets

Materials and reagents

Purification of the β2-AR

Selectivity and system suitability of β2-AR column

Conclusion

Acknowledgments

Eur. J. Med. Chem.

Life Sci.

Anal. Chim. Acta

Anal. Chim. Acta

Fitoterapia

Biochim. Biophys. Acta

Drug Discov. Today

Life Sci.

Bioorg. Med. Chem.

Biochem. Pharmacol.

J. Chromatogr. B

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

Purification of the β₂-AR

Selectivity and system suitability of β₂-AR column