Chemical Diversity of Natural Resources and the Bioactivity of their Constituents

Natural products (NPs) have historically been an important source of lead molecules in drug discovery. However, the interest that the pharmaceutical industry has had in NPs has declined in part because of the lack of compatibility of traditional natural-product extract libraries with high-throughput screenings and the low hit rate. Furthermore, in contrast to the synthetic libraries, compounds from natural sources are likely to have complex structures which slow down the identification process and contribute to problems related to supply and manufacturing. In this paper, we summarise some of the strategies that are being developed in our research unit to address these issues. On one hand, differential screening strategies were established with the aim of identifying dynamically induced NPs from silent biosynthetic pathways in plants and fungi that had been exposed to different stress situations. On the other hand, high-resolution HPLC techniques were optimised for biological and chemical profiling of crude extracts. This led to an integrated platform for rapid and efficient identification of new drug-leads and biomarkers of interest that were based on miniaturised technological approaches and metabolomics

The Potential of Higher Plants as a Source of New Drugs

The plant kingdom is still an untapped reservoir of new molecules with therapeutic potential. A selection of bioactive plant constituents recently discovered are presented with focus on new drugs or lead compounds in an advanced state of development. Obtaining pure new biologically active substances from plants remains a complex task. Biological and chemical screenings are complementary approaches for the rapid detection and isolation of new interesting plant constituents. Biological screening followed by activity-guided fractionation has been successfully used in our laboratories for the discovery of new antifungal metabolites and inhibitors of enzymes involved in the aetiology of prostate hyperplasia. High-performance liquid chromatography (HPLC) coupled to UV spectroscopy (LC/UV), mass spectrometry (LC/MS), and magnetic resonance (LC/NMR) proved to be highly efficient for the chemical screening of crude plant extracts. These hyphenated techniques were extensively used for the investigation of polyphenols with monoamine-oxidase inhibitory (IMAO) properties in Gentianaceae species

Bioactivity-guided isolation of trypanocidal coumarins and dihydro-pyranochromones from selected Apiaceae plant species.

Bioactivity-guided isolation of natural products from plant matrices is widely used in drug discovery. Here, this strategy was applied to identify trypanocidal coumarins effective against the parasite Trypanosoma cruzi, the etiologic agent of Chagas disease (American trypanosomiasis). Previously, phylogenetic relationships of trypanocidal activity revealed a coumarin-associated antichagasic hotspot in the Apiaceae. In continuation, a total of 35 ethyl acetate extracts of different Apiaceae species were profiled for selective cytotoxicity against T. cruzi epimastigotes over host CHO-K1 and RAW264.7 cells at 10 μg/mL. A flow cytometry-based T. cruzi trypomastigote cellular infection assay was employed to measure toxicity against the intracellular amastigote stage. Among the tested extracts, Seseli andronakii aerial parts, Portenschlagiella ramosissima and Angelica archangelica subsp. litoralis roots exhibited selective trypanocidal activity and were subjected to bioactivity-guided fractionation and isolation by countercurrent chromatography. The khellactone ester isosamidin isolated from the aerial parts of S. andronakii emerged as a selective trypanocidal molecule (selectivity index ∼9) and inhibited amastigote replication in CHO-K1 cells, though it was significantly less potent than benznidazole. The khellactone ester praeruptorin B and the linear dihydropyranochromones 3'-O-acetylhamaudol and ledebouriellol isolated from the roots of P. ramosissima were more potent and efficiently inhibited the intracellular amastigote replication at < 10 μM. The furanocoumarins imperatorin, isoimperatorin and phellopterin from A. archangelica inhibited T. cruzi replication in host cells only in combination, indicative of superadditive effects, while alloimperatorin was more active in fractions. Our study reports preliminary structure-activity relationships of trypanocidal coumarins and shows that pyranocoumarins and dihydropyranochromones are potential chemical scaffolds for antichagasic drug discovery

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

Metabolomics in Ecology and Bioactive Natural Products Discovery: Challenges and Prospects for a Comprehensive Study of the Specialised Metabolome

Metabolomics is playing an increasingly prominent role in chemical ecology and in the discovery of bioactive natural products (NPs). The identification of metabolites is a common/central objective in both research fields. NPs have significant biological properties and play roles in multiple chemical-ecological interactions. Classically, in pharmacognosy, their chemical structure is determined after a complex process of isolating and interpreting spectroscopic data. With the advent of powerful analytical techniques such as liquid chromatography-mass spectrometry (LC-MS) the annotation process of the specialised metabolome of plants and microorganisms has improved considerably. In this article, we summarise the possibilities opened by these advances and illustrate how we harnessed them in our own research to automate annotations of NPs and target the isolation of key compounds. In addition, we are also discussing the analytical and computational challenges associated with these emerging approaches and their perspective

Salvia officinalis for Hot Flushes: Towards Determination of Mechanism of Activity and Active Principles

Herbal medicinal products are commonly used in alternative treatment of menopausal hot flushes. In a recent clinical study, Salvia officinalis tincture was found to reduce hot flush frequency and intensity. The aim of the current study was the investigation of the mechanism(s) responsible for the anti-hot flush activity of S. officinalis and determination of its active principle(s). The 66 % ethanolic tincture, as well as the n-hexane, CHCl3, and aqueous ethanolic subextracts obtained from the tincture were studied in vitro for two of the most relevant activities, estrogenicity and selective serotonin reuptake inhibition. Because of an increased risk of menopausal women to suffer from Alzheimerʼs disease, an in vitro acetylcholinesterase inhibition assay was also employed. No activity was observed in the selective serotonin reuptake inhibition or the acetylcholinesterase inhibition assays at the highest test concentrations. The tincture showed no estrogenic effects whereas the aqueous ethanolic subextract exhibited estrogenicity in the ERLUX assay with an EC50 value of 64 µg/mL. Estrogenic activity-guided fractionation of the aqueous ethanolic subextract by a combination of reverse-phase vacuum liquid chromatography and gel chromatography identified luteolin-7-O-glucuronide (EC50 129 µg/mL) as the active component of the vacuum liquid chromatography fraction 4 (EC50 69 µg/mL). Luteolin-7-O-glucoside was identified as the putative estrogenic principle of the most potent minor fraction (7.6.7.6, EC50 0.7 µg/mL) obtained from the initial vacuum liquid chromatography fraction 7 (EC50 3 µg/mL). This study suggests the involvement of common and ubiquitous estrogenic flavonoids in the anti-hot flush effect of Salvia officinalis, a safe and commonly used herbal medicinal product during the menopause