21 research outputs found
Zebrafish Bioassay-guided Microfractionation for the Rapid in vivo Identification of Pharmacologically Active Natural Products
The rapid acquisition of structural and bioactivity information on natural products (NPs) at the sub- milligram scale is key for performing efficient bioactivity-guided isolations. Zebrafish offer the possibility of rapid in vivo bioactivity analysis of small molecules at the microgram
scale – an attractive feature when combined with high-resolution fractionation technologies and analytical methods such as UHPLC-TOF-MS and microflow NMR. Numerous biomedically relevant assays are now available in zebrafish, encompassing most indication areas. Zebrafish also provide
the possibility to screen bioactive compounds for potential hepato-, cardio-, and neurotoxicities at a very early stage in the drug discovery process. Here we describe two strategies using zebrafish bioassays for the high-resolution in vivo bioactivity profiling of medicinal plants, using
either a one-step or a two-step procedure for active compound isolation directly into 96-well plates. The analysis of the microfractions by microflow NMR in combination with UHPLC-TOF-MS of the extract enables the rapid dereplication of compounds and an estimation of their microgram quantities
for zebrafish bioassays. Both the one-step and the two-step isolation procedures enable a rapid estimation of the bioactive potential of NPs directly from crude extracts. In summary, we present an in vivo , microgram-scale NP discovery platform combining zebrafish bioassays with microscale
analytics to identify, isolate and evaluate pharmacologically active NPs
Advanced strategies for the study of bioactive plant constituents and co-culture induced fungal metabolites
This thesis aims at investigating novel and rational strategies to identify secondary metabolites from plant or microbial origin by targeted isolation; either guided by HPLC-bioactivity profiling or by metabolomics. Firstly, a microfractionation strategy was elaborated for mass-limited extracts that is compatible with the detection and identification of the purified compounds by microflow NMR as well as in vivo zebrafish bioassays. This approach revealed several flavanoids that explain the antiangiogenic activity of the Rhynchosia viscosa extract. Secondly, strategies were developed for the analysis of microorganism co-cultures. Microfractionation led to the isolation of a co-culture induced compound in the low microgram range from the co-culture of Acremonium strictum and Fusarium oxysporum. For the co-culture of Hohenbuehelia reniformis and Fusarium solani, multivariate data analysis highlighted metabolites responsible for the differences between pure culture and co-culture extracts. A two-step chromatographic purification permitted the targeted isolation of highlighted metabolites – u.a. several novel and anti-Fusarium quinones
On-line and At-line Liquid Chromatography Nuclear Magnetic Resonance and Related Micro-Nuclear Magnetic Resonance Methods in Natural Product Analysis
Nuclear magnetic resonance spectroscopy (NMR) is essential for the de novo structure identification of natural products. This technique can be directly or indirectly hyphenated to high performance liquid chromatography (HPLC) leading to efficient LC-NMR platforms. NMR is, however, a very insensitive detection method compared to mass spectrometry and several strategies for its efficient hyphenation with HPLC have been developed. This includes on-line LC-NMR, SPE-NMR, or other at-line approaches involving microflow probes or microtubes in combination with cryoprobes. All of these various technologies and their potentials and limitations in the field of natural product research are described. Recent applications involving other complementary detection methods such as LC-MS or biological HPLC profiling illustrate its utilization
Advanced Methods for Natural Product Drug Discovery in the Field of Nutraceuticals
Advances in analytical methods and bioassay development have helped to push forward the research in natural products. In plant extracts and nutraceuticals, bioactive compounds are part of a complex mixture. The development of high-resolution methods related to HPLC for both chemical and biological profiling has significantly increased the efficiency of classical bioactivity-guided fractionation procedures. Furthermore, the level of sensitivity obtained by these methods give the possibility to work with few micrograms of compound. This represents a key advantage for rapid localisation of the biological activity and subsequent identification of the compounds of interest. The same methods are also used to study the extracts from a metabolomic view point. The possibility to study them as a whole can highlight synergetic effects, which are likely to occur in plant extracts and nutraceuticals. In this paper, the main trends are summarised and the developments made in our laboratory on profiling crude extracts with UHPLC-TOF-MS, natural product identification at the microgram level using microflow NMR and integration of these methods with biological evaluation are highlighted
Metabolite induction via microorganism co-culture: A potential way to enhance chemical diversity for drug discovery
Microorganisms have a long track record as important sources of novel bioactive natural products, particularly in the field of drug discovery. While microbes have been shown to biosynthesize a wide array of molecules, recent advances in genome sequencing have revealed that such organisms have the potential to yield even more structurally diverse secondary metabolites. Thus, many microbial gene clusters may be silent under standard laboratory growth conditions. In the last ten years, several methods have been developed to aid in the activation of these cryptic biosynthetic pathways. In addition to the techniques that demand prior knowledge of the genome sequences of the studied microorganisms, several genome sequence-independent tools have been developed. One of these approaches is microorganism co-culture, involving the cultivation of two or more microorganisms in the same confined environment. Microorganism co-culture is inspired by the natural microbe communities that are omnipresent in nature. Within these communities, microbes interact through signaling or defense molecules. Such compounds, produced dynamically, are of potential interest as new leads for drug discovery. Microorganism co-culture can be achieved in either solid or liquid media and has recently been used increasingly extensively to study natural interactions and discover new bioactive metabolites. Because of the complexity of microbial extracts, advanced analytical methods (e.g., mass spectrometry methods and metabolomics) are key for the successful detection and identification of co-culture-induced metabolites. This review focuses on co-culture studies that aim to increase the diversity of metabolites obtained from microbes. The various strategies are summarized with a special emphasis on the multiple methods of performing co-culture experiments. The analytical approaches for studying these interaction phenomena are discussed, and the chemical diversity and biological activity observed among the induced metabolites are described
The 40th Swiss Group for Mass Spectrometry Meeting: Conference Report
The Swiss Group for Mass Spectrometry (SGMS) is a society dedicated to fostering collaboration among scientists across diverse mass spectrometry fields, ranging from gas-phase ion chemistry to life sciences. SGMS has a strong commitment to educating and supporting the next generation of mass spectrometrists pursuing MSc and PhD degrees. Our membership comprises about two hundred individuals, predominantly from Switzerland, representing academia, industry, and regulatory bodies.[1] SGMS is an Associate Member Society of the Swiss Chemical Society (SCS)
<i>De Novo</i> Production of Metabolites by Fungal Co-culture of <i>Trichophyton rubrum</i> and <i>Bionectria ochroleuca</i>
The co-cultivation of fungi has recently
been described as a promising
strategy to induce the production of novel metabolites through possible
gene activation. A large screening of fungal co-cultures in solid
media has identified an unusual long-distance growth inhibition between <i>Trichophyton rubrum</i> and <i>Bionectria ochroleuca</i>. To study metabolite induction in this particular fungal interaction,
differential LC-MS-based metabolomics was performed on pure strain
cultures and on their co-cultures. The comparison of the resulting
fingerprints highlighted five <i>de novo</i> induced compounds,
which were purified using software-oriented semipreparative HPLC-MS.
One metabolite was successfully identified as 4″-hydroxysulfoxy-2,2″-dimethylthielavin
P (a substituted trimer of 3,5-dimethylorsellinic acid). The nonsulfated
form, as well as three other related compounds, were found in the
pure strain culture of <i>B. ochroleuca</i>