A robust and miniaturized screening platform to study natural products affecting metabolism and survival in Caenorhabditis elegans

Abstract

In this study a robust, whole organism screening based on Caenorhabditis elegans is presented for the discovery of natural products (NP) with beneficial effects against obesity and age-related diseases. Several parameters of the elaborated workflow were optimized to be adapted for probing multicomponent mixtures combining knowledge from traditional medicine and np chemistry by generating optimized small-scale extracts considering scarcity of the natural source, solubility issues, and potential assay interferences. The established miniaturized assay protocol allows for in vivo probing of small amounts of even complex samples (~ 1 mg) to test their ability to increase the nematodes' survival time and the suppression of fat accumulation assessed by nile red staining as hall marks of "healthy aging". The workflow was applied on 24 herbal and fungal materials traditionally used against symptoms of the metabolic syndrome and revealed promising results for the extracts of Gardenia jasminoides fruits and the sclerotia from Inonotus obliquus. Tested at 100 µg/mL they were able to significantly reduce the Nile red fluorescence and extend the 50% survival rate (DT 50) compared to the control groups. This phenotype-directed in vivo approach opens up new horizons for the selection of natural starting materials and the investigation of their active principles as fast drug discovery tool with predictive value for human diseases. Caenorhabditis elegans (Maupas, 1900), a 1 mm sized roundworm, is a popular model organism in almost all areas of modern biology. It can be maintained at low cost, has a short reproductive cycle of three days with a large brood size of 300 progenies per hermaphrodite worm and a transparent body comprising exactly 959 somatic cells 1. In recent years it has been increasingly used as a model organism for drug screenings 2-7. The fundamental idea behind is that basic molecular processes which are causal for the development of diseases including aging processes are conserved in the animal kingdom. Indeed C. elegans shares many similarities with humans such as autophagy, mitochondrial regulation, apoptosis, proteostasis, energy control, fat-storage, stress response systems and neuronal regeneration 8-16. A recent meta-analysis estimated that 41.7% of the protein-coding genes in C. elegans have orthologs in humans 17. In this light, screening for substances beneficial to a disease phenotype in C. elegans can have important predictive value also for human diseases 18, 19. The simplicity and tractability of the worm compared to classical mammal models represents a large advantage. Its small size makes it amenable to whole organism screening in microtiter plates for medium/high-throughput screening with little consumption of materials and sample 20. This possibility is particularly helpful for drug discovery from natural sources, which is often impeded by scarcity of natural starting materials, and even more relevant for their isolates which require tedious isolation or synthesizing efforts 21-24. There is an in...

Workflow for Segmentation of Caenorhabditis elegans from Fluorescence Images for the Quantitation of Lipids

“…Both organisms were obtained from the Caenorhabditis Genetics Center (University of Minnesota). Details of the miniaturized Nile red assay in C. elegans including the composition of all media and reagents have been published recently [21]. Briefly, hermaphrodite animals were maintained on nematode growth medium (NGM) agar plates seeded with 20 µg of OP50 at 16 • C as described by Stiernagle [36].…”

“…The applicability of the presented method was outlined using positive controls fluoxetine and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) as an application example in [21]. Fluoxetine (F-132) and AICAR (A9978) were obtained from Sigma Aldrich with a purity of ≥98%.…”

“…We have recently established a miniaturized fat accumulation assay in 96-well plates based on the staining of lipid compartments with Nile red [21]. For the efficient quantitation of lipid-derived fluorescence of worms, an image processing workflow capable of segmenting worms from fluorescence microscopy images was necessary.…”

Peucedanum ostruthium Inhibits E-Selectin and VCAM-1 Expression in Endothelial Cells through Interference with NF-κB Signaling

“…Tannin-rich extracts were further subjected to tannin depletion to avoid any unspecific assay interferences. Protocols for the generation of tannin-depleted samples have been described previously [12,13] and successfully applied in several studies [11,[14][15][16]. For this process, the dried CH 2 Cl 2 -MeOH extracts were restored in 4-mL MeOH, loaded onto a 3-mL solid-phase extraction cartridge (Phenomenex, Torrance, CA, USA; AH0-7001) filled with polyamide gel (Carl Roth, Karlsruhe, Germany; CC-6; 900 mg) and washed two times with MeOH.…”

Fruit of Gardenia jasminoides Induces Mitochondrial Activation and Non-Shivering Thermogenesis through Regulation of PPARγ

“…Recent studies also suggest the possibility of GJF as an anti-obesity agent. For instance, Park et al reported that the ethanol extract of GJF inhibited adipogenesis in white-induced 3T3-L1 cells [ 15 ], and Zwirchmayr et al showed that methylene chloride and methanol extracts of GJF reduced lipid accumulation in Caenorhabditis elegans [ 16 ]. However, whether GJF has an effect on non-shivering thermogenesis (NTS) is unknown.…”