Bioactive Agents from Natural Sources: Trends in Discovery and Application - Advances in Biochemical Engineering Biotechnology - page 119

4.1.1
Physico-Chemical Screening
Mycelium extracts,culture filtrates,or crude extracts ofmicrobial broths as well
as samples obtained from plant and animal extraction can be subjected to
standardized reversed-phase HPLC by making use of various coupling techni-
ques. Most common is HPLC coupled to a multi-wavelength UV/VI S -monitor
(diode array detection,DAD) [147].Comparison ofthe data (retention time and
UV/VI S -spectra) to reference substances acts as selection criteria. However,
success ofthis strategy depends upon the amount and quality ofpure references
in the database.Based on the correlation to UV/VIS monitoring the HPLC-DAD
screening is well suitable for screening towards metabolites which bear signi-
ficant chromophores. In combination with the efficient separation via HPLC
this screening procedure can advantageously be applied to plant material which
exhibits numerous colored compounds. The HPLC-DAD screening has al-
ready been successfully applied to natural products screening and led to the dis-
covery of several new metabolites like the naphthoquinone juglomycin Z [148],
naphthgeranine F [149], the antibiotically active fatty acid ( E )-4-oxonon-2-
enoic acid [150], the insecticidal acting NK374200 [151], or the quinoxaline
group metabolite echinoserine [152].
The data obtained from HPLC-DAD analysis are often helpful in dereplica-
tion,e.g.during high-throughput biological screening programs.However,the
dependence on a UV/VIS detectable chromophor in the metabolite to be analy-
zed limits its possible application in principal.Therefore,alternative detection
methods like mass spectrometry (LC-MS) [153,154],or nuclear magnetic reso-
nance (LC-NMR) [155] have been applied.In a further concept,analytical data
from HPLC-DAD can advantageously be supplemented and combined with data
from HPLC-ESI-MS.An example for a successful screening is the discovery of
the polyol macrolides of the kanchanamycin group [156,157].
4.1.2
Chemical Screening
In order to apply chemical screening on TLC starting from microbial cultures in
a reproducable way,standardized procedures for sample preparation and at least
50-fold concentration are required. The obtained concentrates from both, the
mycelium and the culture filtrate are analyzed by applying a defined amount to
high-performance thin-layer chromatography (HPTLC) silica-gel plates which
then are chromatographed using different solvent systems. In a next step the
metabolite pattern of each strain is analyzed by making use of visual detection
(colored substances), UV-extinction/fluorescence, and colorization reactions
obtained by staining with different reagents (e.g. anisaldehyde/sulfuric acid-,
naphthoresorcin/sulfuric acid-, orcinol-, blue terazolium-, and Ehrlich's
reagent).The advantage of these reagents lies in the broad structural spectrum
of metabolites stained.
This procedure mainly focuses on the chemical behavior and reactivity ofthe
components and gives a good visualization of the secondary metabolite pattern