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...

Chemometric methods and correlation of spectroscopic or spectrometric data with bioactivity results are known to improve dereplication in classical bio-guided isolation approaches. However, in drug discovery from natural sources the isolation of bioactive constituents from a crude extract containing close structural analogues remains a significant challenge. This study is a 1 H NMR-MS workflow named ELINA ( Eli citing N ature’s A ctivities) which is based on statistical heterocovariance analysis (HetCA) of 1 H NMR spectra detecting chemical features that are positively (“hot”) or negatively (“cold”) correlated with bioactivity prior to any isolation. ELINA is exemplified in the discovery of steroid sulfatase (STS) inhibiting lanostane triterpenes (LTTs) from a complex extract of the polypore fungus Fomitopsis pinicola .

Peucedanum ostruthium (L.) Koch, commonly known as masterwort, has a longstanding history as herbal remedy in the Alpine region of Austria, where the roots and rhizomes are traditionally used to treat disorders of the gastrointestinal and respiratory tract. Based on a significant NF-κB inhibitory activity of a P. ostruthium extract (PO-E), this study aimed to decipher those constituents contributing to the observed activity using a recently developed biochemometric approach named ELINA (Eliciting Nature’s Activities). This -omics tool relies on a deconvolution of the multicomponent mixture, which was employed by generating microfractions with quantitative variances of constituents over several consecutive fractions. Using an optimized and single high-performance counter-current chromatographic (HPCCC) fractionation step 31 microfractions of PO-E were obtained. 1H NMR data and bioactivity data from three in vitro cell-based assays, i.e., an NF-ĸB reporter-gene assay and two NF-κB target-gene assays (addressing the endothelial adhesion molecules E-selectin and VCAM-1) were collected for all microfractions. Applying heterocovariance analyses (HetCA) and statistical total correlation spectroscopy (STOCSY), quantitative variances of 1H NMR signals of neighboring fractions and their bioactivities were correlated. This revealed distinct chemical features crucial for the observed activities. Complemented by LC-MS-CAD data this biochemometric approach differentiated between active and inactive constituents of the complex mixture, which was confirmed by NF-κB reporter-gene testing of the isolates. In this way, four furanocoumarins (imperatorin, ostruthol, saxalin, and 2’-O-acetyloxypeucedanin), one coumarin (ostruthin), and one chromone (peucenin) were identified as NF-κB inhibiting constituents of PO-E contributing to the observed NF-ĸB inhibitory activity. Additionally, this approach also enabled the disclose of synergistic effects of the PO-E metabolites imperatorin and peucenin. In sum, prior to any isolation an early identification of even minor active constituents, e.g. peucenin and saxalin, ELINA enables the targeted isolation of bioactive constituents and, thus, to effectively accelerate the NP-based drug discovery process.

Twenty natural remedies traditionally used against different inflammatory diseases were probed for their potential to suppress the expression of the inflammatory markers E-selectin and VCAM-1 in a model system of IL-1 stimulated human umbilical vein endothelial cells (HUVEC). One third of the tested extracts showed in vitro inhibitory effects comparable to the positive control oxozeaenol, an inhibitor of TAK1. Among them, the extract derived from the roots and rhizomes of Peucedanum ostruthium (i.e., Radix Imperatoriae), also known as masterwort, showed a pronounced and dose-dependent inhibitory effect. Reporter gene analysis demonstrated that inhibition takes place on the transcriptional level and involves the transcription factor NF-κB. A more detailed analysis revealed that the P. ostruthium extract (PO) affected the phosphorylation, degradation, and resynthesis of IκBα, the activation of IKKs, and the nuclear translocation of the NF-κB subunit RelA. Strikingly, early effects on this pathway were less affected as compared to later ones, suggesting that PO may act on mechanism(s) that are downstream of nuclear translocation. As the majority of cognate NF-κB inhibitors affect upstream events such as IKK2, these findings could indicate the existence of targetable signaling events at later stages of NF-κB activation.

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In an screening for anti-influenza agents from European polypores, the fruit body extract of dose-dependently inhibited the cytopathic effect of the H3N2 influenza virus A/Hong Kong/68 (HK/68) in Madin Darby canine kidney cells with a 50% inhibitory concentration (IC) of 15 µg/mL, a noncytotoxic concentration. After a chromatographic work-up, eight lanostane triterpenes (1: -8: ) were isolated and their structures were elucidated based on high-resolution electrospray ionization mass spectrometry analyses, and one- and two-dimensional nuclear magnetic resonance experiments. Constituents 1: (gloeophyllin K) and 2: (gloeophyllin L) are reported here for the first time, and compounds 5: , 7: , and 8: have not been described for the investigated fungal material so far. The highest activity was determined for trametenolic acid B (3: ) against HK/68 and the 2009 pandemic H1N1 strain A/Jena/8178/09 with IC values of 14 and 11 µM, respectively. In a plaque reduction assay, this compound was able to bind to cell-free viruses and to neutralize their infectivity.

Based on the traditional use and scientific reports on the anti-inflammatory potential of red sandalwood, i.e., the heartwood of Pterocarpus santalinus L., we investigated its activity in a model of IL-1 stimulated endothelial cells. Endothelial cells were stimulated with IL-1 with or without prior incubation with a defined sandalwoodextract (PS), and analyzed for the expression of selected pro-inflammatory genes. The activity of NF-κB, a transcription factor of central importance for inflammatory gene expression was assessed by reporter gene analysis, Western blotting of IκBα, and nuclear translocation studies. In addition, microarray studies were performed followed by verification of selected genes by qPCR and supplemented by bioinformatics analysis. Our results show that PS is able to suppress the induction of E-selectin and VCAM-1, molecules that mediate key steps in the adhesion of leukocytes to the endothelium. It also suppressed the activity of an NF-κB reporter, IκBα phosphorylation and degradation, and the nuclear translocation of NF-κB RelA. In contrast, it stimulated JNK phosphorylation indicating the activation of the JNK signaling pathway. Gene expression profiling revealed that PS inhibits only a specific subset of IL-1 induced genes, while others remain unaffected. Most strongly suppressed genes were the signal transducer TRAF1 and the chemokine CX3CL1, whereas IL-8 was an example of a non-affected gene. Notably, PS also stimulated the expression of certain genes, including ones with negative regulatory function, e.g., members of the NR4A family, the mRNA destabilizing protein TTP as well as the transcription factors ATF3 and BHLHB40. These results provide mechanistic insight into the anti-inflammatory activity of PS, and suggest that it acts through the interplay of negative and positive regulators to achieve a differential inhibition of inflammatory gene expression.

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