Editorial
Received: 12 December 2016, Accepted: 12 December 2016
(wileyonlinelibrary.com) DOI 10.1002/pca.2675
Bioassay-coupled Chromatography: Challenges
and Applications in Natural Product Research
One of the major challenges in drug discovery from natural
sources is the identification of those compounds within these
complex mixtures which are closely related to the observed bio-
logical activities. For this purpose, usually bioassay-guided isola-
tion strategies are employed which can be a tedious and time
consuming procedure, triggering research for methods to speed
up such processes. During recent years two main developments
can be observed. On the one hand, metabolomic analysis of ex-
tracts by high-throughput high resolution mass spectrometry
(MS) coupled to liquid chromatography or by NMR pattern recog-
nition is connected with results from bioassays by quantitative
structure–activity relationship (QSAR) analysis. This can be
regarded as an indirect method and needs further evaluation of
identified markers in the respective assays after isolation. On the
other hand, hyphenation with bioassays added an exciting dimen-
sion to chromatography and enabled identification of bioactive
compounds in complex mixtures without prior isolation of pure
substances. This special issue is dedicated to explore recent devel-
opments in bioassay-coupled chromatography and its application
in natural products analysis and activity screening.
Thin-layer chromatography (TLC) and its higher developed auto-
mated and reproducible variant HPTLC can be regarded as the most
widely applied chromatographic method coupled to bioassays. Al-
though less potent concerning peak resolution and speed com-
pared to ultra-high-performance liquid chromatography (UHPLC),
the possibility to use the developed TLC plate carrying the sepa-
rated natural products after evaporation of the often biotoxic sol-
vents as playground for diverse reactions to detect biological
activities has attracted many research groups. Bräm and Wolfram
thoroughly discussed in their review the recent developments in
TLC-based enzyme assays. The review provides a systematic update
of the recently published approaches to methodological optimisa-
tion for each of the so far reported TLC-enzyme inhibition assays.
The review discusses the improvements and pitfalls of the method-
ology and discusses future applications as well as the need for fur-
ther research in this field. The hyphenation of TLC and MS for
elucidation of structural information of the positive enzyme inhibi-
tors as well as control experiments for avoidance of artefacts seem
to be the key factors for a broad applicability of these efficient com-
bined analytical and activity screening methods.
Currently, the search for inhibitors of tyrosinase has gained much
attention. Their potential medical and cosmetic applications include
hyperpigmentation disorders such as chloasma or solar/senile
lentigines resulting from melanin overproduction. Beyond such
use tyrosinase inhibitors are evaluated as anti-browning agents in
food production as well as alternative agents for insect control.
Zhough et al. present an improved TLC-based tyrosinase assay
which enables qualitative and quantitative estimation of the en-
zyme inhibition reaction. The substrate L-tyrosine was replaced by
the better water soluble L-3,4-dihydroxyphenylalanin (L-DOPA),
Phytochem. Anal. 2017 Copyright © 2017 John Wiley & Sons, Ltd.
Published online in Wiley Online Library
quantification was performed through densitometry and method
optimisation resulted in a detection limit of 1 ng kojic acid and a re-
duced enzyme consumption of 75 U/mL. Whereas most TLC-based
enzyme assays are employing silica as stationary phase, Garcia et al.
developed a tyrosinase inhibition assay on reversed phase TLC
plates offering a complementary chromatographic selectivity.
Chaita et al. used two methods of data set generation and multivar-
iate data analysis approaches to analyse tyrosinase inhibiting frac-
tions from Morus alba L. separated by HPTLC after pre-
fractionation with centrifugal partition chromatography. Although
not performing the enzyme assay directly on the TLC plate this study
represents a valuable example of connecting data mining with
HPTLC for tracing bioactive compounds.
Treatment of diabetes-2 with α-glucosidase inhibitors in combi-
nation with an appropriate diet is a current therapeutic option to
cope with the ever increasing prevalence of this disease. Natural
products play an important role as α-glucosidase inhibitors, which
is also illustrated by the study of Theiler et al. who could isolate α-
glucosidase inhibitors from Justicia secunda Vahl using HPTLC
bioautography.
Column liquid chromatographic techniques still cover the ma-
jority of analytical tasks in natural products analysis. Due to limited
reaction time available, linking (U)HPLC to enzyme assays is mainly
performed by microfractionation followed by the respective assays
carried out in microtiter plate format. Zwick et al. not only used
HPLC for microfractionation in combination with an assay to de-
tect inhibitors of histone deacetylase (HDAC) isoforms, promising
targets in cancer treatment, but also employed UHPLC-ESI-MS/
MS for analysing the enzyme reaction and by that avoiding inter-
ference of background fluorescence in the HDAC assay.
As an example that direct on-line coupling of liquid chromatog-
raphy to bio-assays is feasible the study of Opitz et al. can serve. A
recently developed on-line ABTS assay coupled to high perfor-
mance size exclusion chromatography was used to study the anti-
oxidants in coffee brews.
The papers collected in this special issue shed light on a few
facets of the potential of hyphenation of chromatography to bioas-
says for bioactive compound identification. It should also serve as a
stimulus to encourage research into highly demanded methods to
accelerate the process from detection of a biological activity in a
crude extract to the identification of the bioactivity markers.
Franz Bucar
Institute of Pharmaceutical Sciences, University of Graz,
Universitätsplatz 4 8010 Graz Austria
franz.bucar@uni-graz.at
Evelyn Wolfram
ZHAW Life Sciences und Facility Management
Institute of Chemistry and Biotechnology Einsiedlerstrasse 31 8820
Wädenswil, Switzerland