Download Discovery of xanthine oxidase inhibitors from a complex mixture

Anal Bioanal Chem (2014) 406:1975–1984
DOI 10.1007/s00216-013-7612-8
RESEARCH PAPER
Discovery of xanthine oxidase inhibitors from a complex
mixture using an online, restricted-access material coupled
with column-switching liquid chromatography
with a diode-array detection system
De-qiang Li & Jing Zhao & Shao-ping Li & Qing-wen Zhang
Received: 22 October 2013 / Revised: 25 December 2013 / Accepted: 30 December 2013 / Published online: 9 February 2014
# Springer-Verlag Berlin Heidelberg 2014
Abstract To find potential lead compounds for antigout drug
discovery, an automated online, restricted-access material
coupled with column-switching liquid chromatography with
a diode-array detection (RAM–LC–DAD) system was developed for screening of xanthine oxidase (XO) inhibitors and
their affinity rankings in complex mixtures. The system was
first evaluated by analyzing a mixture of six compounds
with known inhibition of XO. Nonspecific binding to the
denatured XO was investigated and used as the control for
screening. Subsequently, the newly developed system was
applied to screening of a natural product, Oroxylum indicum
extract, and four compounds which could specifically interact
with XO were found and identified as oroxin B, oroxin A,
baicalin, and baicalein. The results were verified by a competitive binding test using the known competitive inhibitor
allopurinol and were further validated by an inhibition assay
in vitro. The online RAM–LC–DAD system developed was
shown to be a simple and effective strategy for the rapid
screening of bioactive compounds from a complex mixture.
Keywords Xanthine oxidase inhibitors . Restricted-access
material . Column switching . Liquid chromatography
Abbreviations
DAD Diode-array detection
HPLC High-performance liquid chromatography
IC50
Half-maximal inhibitory concentration
LC
Liquid chromatography
MS
Mass spectrometry
D.<q. Li : J. Zhao (*) : S.<p. Li (*) : Q.<w. Zhang
State Key Laboratory of Quality Research in Chinese Medicine,
Institute of Chinese Medical Sciences, University of Macau,
Macao, China
e-mail: [email protected]
e-mail: [email protected]
RAM
RSD
SEC
SPE
TFC
XO
Restricted-access material
Relative standard deviation
Size-exclusion chromatography
Solid-phase extraction
Turbulent flow chromatography
Xanthine oxidase
Introduction
Gout, a fairly common metabolic disorder characterized by
chronic hyperuricemia, is markedly increasing in prevalence
in the USA as well as other countries with established and
emerging economies, and has become a health problem
worldwide [1, 2]. The population with gout in the USA has
been estimated at 6.1 million, and studies in the UK have
reported a prevalence approaching 7 % [3]. Global studies
have found an increase in mean serum urate levels in both
genders during the past four decades [4]. Inhibition of xanthine oxidase (XO) is recognized as the major treatment of
gout and its complications [5]. At present, allopurinol used as
a primary XO inhibitor is being prescribed to prevent gout
attacks, but it was claimed to have potentially serious side
effects such as hypersensitivity syndrome [6]. Therefore, considerable efforts are being made to find alternative inhibitors
from natural sources because of their chemical diversity and
excellent efficacy as well as low side effects [7]. However, it is
a great challenge to discover active compounds from complex
matrices, commonly involving numerous repeated chemical
analysis, sample fractionation, and biological screening.
Therefore, there is an urgent need to develop rapid and simple
methods for screening of XO inhibitors from natural products.
At present, high-throughput screening of bioactive compounds from complex mixtures mainly relies on the binding