Identification of C. elegans lipid lowering constituents form chaga by correlation of ¹H NMR spectra with phenotypic screening

Natural products and phenotype directed screening in animal models have been prolific fields for the discovery of innovative small molecule drugs – also in the field of metabolic disease [1]–[2]. Since the screening of extracts and natural products (NP) in classical obesity rodent models is hampered by several disadvantages, e.g. financial efforts, legal and ethical considerations and the requirement of large quantities of test materials, we established an obesity model in the nematode Caenorhabditis elegans (C. elegans). The C. elegans obesity assay is based on lipid staining of the mutant strain SS104 with the fluorescent dye Nile Red. It can be performed with little sample amounts in 96-well plates with medium throughput. An ethanolic extract of chaga, i.e. the fruit bodies of the mushroom Inonotus obliquus (Ach. ex Pers.) Pilát, significantly inhibited the fat accumulation in C. elegans at 10 μg/mL, without having a negative effect on the nematodes’ lifespan and was therefore selected for further mycochemical investigations. The extract was separated via flash chromatography into 37 micro-fractions with quantitative variances of constituents over several consecutive fractions. The effect on fat accumulation was determined for each fraction and the increases and decreases of bioactivity were correlated with the fractions’ ¹H NMR spectra following a recently developed biochemometric approach named ELINA (Eliciting Nature’s Activities) [3]. Together with UPC²-ELSD-QDa data we were able to pinpoint several constituents and their chemical features responsible for the observed phenotypic effect, which enabled their targeted isolation.

Acknowledgement

We thank Dr. Agnieszka Kowalska, Department of Pharmacognosy, University of Vienna, for her excellent technical support, and Pakuso LLC, Lieto, Finland for providing the mushroom material.

• References

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