Antiprotozoal Activity of Plants Used in the Management of Sleeping Sickness in Angola and Bioactivity-Guided Fractionation of Brasenia schreberi J.F.Gmel and Nymphaea lotus L. Active against T. b. rhodesiense.

Folk medicine is widely used in Angola, even for human African trypanosomiasis (sleeping sickness) in spite of the fact that the reference treatment is available for free. Aiming to validate herbal remedies in use, we selected nine medicinal plants and assessed their antitrypanosomal activity. A total of 122 extracts were prepared using different plant parts and solvents. A total of 15 extracts from seven different plants exhibited in vitro activity (>70% at 20 µg/mL) against Trypanosoma brucei rhodesiense bloodstream forms. The dichloromethane extract of Nymphaea lotus (leaves and leaflets) and the ethanolic extract of Brasenia schreberi (leaves) had IC50 values ≤ 10 µg/mL. These two aquatic plants are of particular interest. They are being co-applied in the form of a decoction of leaves because they are considered by local healers as male and female of the same species, the ethnotaxon "longa dia simbi". Bioassay-guided fractionation led to the identification of eight active molecules: gallic acid (IC50 0.5 µg/mL), methyl gallate (IC50 1.1 µg/mL), 2,3,4,6-tetragalloyl-glucopyranoside, ethyl gallate (IC50 0.5 µg/mL), 1,2,3,4,6-pentagalloyl-ß-glucopyranoside (IC50 20 µg/mL), gossypetin-7-O-ß-glucopyranoside (IC50 5.5 µg/mL), and hypolaetin-7-O-glucoside (IC50 5.7 µg/mL) in B. schreberi, and 5-[(8Z,11Z,14Z)-heptadeca-8,11,14-trienyl] resorcinol (IC50 5.3 µg/mL) not described to date in N. lotus. Five of these active constituents were detected in the traditional preparation. This work provides the first evidence for the ethnomedicinal use of these plants in the management of sleeping sickness in Angola.

Identification of Potential Antiseizure Agents in Boswellia sacra using In Vivo Zebrafish and Mouse Epilepsy Models.

The resin of the tree Boswellia sacra Flueck. (synonym: B. carterii; Burseraceae), also known as "frankincense", is a traditional remedy used for central nervous system disorders in East Africa. Here we report the evaluation of its antiseizure activity in zebrafish and mouse epilepsy models to identify novel antiseizure compounds. The resin was extracted by solvents of increasing polarity. The hexane extract demonstrated the strongest antiseizure activity and was therefore subjected to bioactivity-guided isolation, which leaded to the isolation of eight terpene derivatives. A new prenylbicyclogermacrene derivative (2) was isolated along with seven other compounds (1, 3-8). Among them, the triterpene ß-boswellic acid (5) showed the strongest activity and reduced 90% of pentylenetetrazole (PTZ)-induced seizures at 100 µg/mL. In parallel to B. sacra, a commercial extract of Boswellia serrata was also evaluated and showed moderate bioactivity (45% reduction at 30 µg/mL). The extract of B. serrata was subjected to targeted isolation of other boswellic acid derivatives (9-13), which were evaluated for antiseizure activity in comparison with 5. In the whole series, ß-boswellic acid (5) was the most active (60% reduction at 200 µM), and its potency was also confirmed with its purchased standard (S5). Pure nanoparticles of S5 and a commercially formulated extract of B. serrata were tested in a PTZ-kindling mouse seizure model. This notably revealed that the S5 administration reduced seizures by 50% in this mouse model, which was consistent with its detection and quantification in plasma and brain samples. This study and the preclinical evaluation performed indicate that ß-boswellic acid, common to various species of Boswellia, has some potential as an antiseizure agent.

Phosphatidylcholines from Pieris brassicae eggs activate an immune response in Arabidopsis.

Recognition of conserved microbial molecules activates immune responses in plants, a process termed pattern-triggered immunity (PTI). Similarly, insect eggs trigger defenses that impede egg development or attract predators, but information on the nature of egg-associated elicitors is scarce. We performed an unbiased bioactivity-guided fractionation of eggs of the butterfly Pieris brassicae. Nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry of active fractions led to the identification of phosphatidylcholines (PCs). PCs are released from insect eggs, and they induce salicylic acid and H2O2 accumulation, defense gene expression and cell death in Arabidopsis, all of which constitute a hallmark of PTI. Active PCs contain primarily C16 to C18-fatty acyl chains with various levels of desaturation, suggesting a relatively broad ligand specificity of cell-surface receptor(s). The finding of PCs as egg-associated molecular patterns (EAMPs) illustrates the acute ability of plants to detect conserved immunogenic patterns from their enemies, even from seemingly passive structures such as eggs.

Metabolite Profiling of Javanese Ginger Zingiber purpureum and Identification of Antiseizure Metabolites via a Low-Cost Open-Source Zebrafish Bioassay-Guided Isolation.

The rhizomes of Zingiber purpureum, "Bangle", were investigated for its antiseizure properties using a streamlined and cost-effective zebrafish screening strategy and a mouse epilepsy assay. Its hexane extract demonstrated strong antiseizure activity in zebrafish epilepsy assay and was, therefore, selected for bioactivity-guided fractionation. Twelve compounds (1-12) were isolated, and two bioactive phenylbutenoids, trans- (11) and cis-banglene (12), reduced up to 70% of pentylenetetrazole (PTZ)-induced seizures. These compounds showed moderate activity against PTZ-induced seizures in a mouse epilepsy assay. To understand the specificity of Z. purpureum active compounds, its chemical profile was compared to that of Z. officinale. Their composition was assessed by differential metabolite profiling visualized by a molecular network, which revealed only vanillin derivatives and terpenoids as common metabolites and gave a comprehensive view of Z. purpureum composition. This study demonstrates the efficacy of a streamlined zebrafish epilepsy assay, which is therefore suitable for routine screening in phytochemistry laboratories.

Antiseizure potential of the ancient Greek medicinal plant Helleborus odorus subsp. cyclophyllus and identification of its main active principles.

ETHNOPHARMACOLOGICAL RELEVANCE: Ethnopharmacological data and ancient texts support the use of black hellebore (Helleborus odorus subsp. cyclophyllus, Ranunculaceae) for the management and treatment of epilepsy in ancient Greece. AIM OF THE STUDY: A pharmacological investigation of the root methanolic extract (RME) was conducted using the zebrafish epilepsy model to isolate and identify the compounds responsible for a potential antiseizure activity and to provide evidence of its historical use. In addition, a comprehensive metabolite profiling of this studied species was proposed. MATERIALS AND METHODS: The roots were extracted by solvents of increasing polarity and root decoction (RDE) was also prepared. The extracts were evaluated for antiseizure activity using a larval zebrafish epilepsy model with pentylenetetrazole (PTZ)-induced seizures. The RME exhibited the highest antiseizure activity and was therefore selected for bioactivity-guided fractionation. Isolated compounds were fully characterized by NMR and high-resolution tandem mass spectrometry (HRMS/MS). The UHPLC-HRMS/MS analyses of the RME and RDE were used for dereplication and metabolite profiling. RESULTS: The RME showed 80% inhibition of PTZ-induced locomotor activity (300 µg/ml). This extract was fractionated and resulted in the isolation of a new glucopyranosyl-deoxyribonolactone (1) and a new furostanol saponin derivative (2), as well as of 20-hydroxyecdysone (3), hellebrin (4), a spirostanol glycoside derivative (5) and deglucohellebrin (6). The antiseizure activity of RME was found to be mainly due to the new furostanol saponin (2) and hellebrin (4), which reduced 45% and 60% of PTZ-induced seizures (135 µM, respectively). Besides, the aglycone of hellebrin, hellebrigenin (S34), was also active (45% at 7 µM). To further characterize the chemical composition of both RME and RDE, 30 compounds (A7-33, A35-37) were annotated based on UHPLC-HRMS/MS metabolite profiling. This revealed the presence of additional bufadienolides, furostanols, and evidenced alkaloids. CONCLUSIONS: This study is the first to identify the molecular basis of the ethnopharmacological use of black hellebore for the treatment of epilepsy. This was achieved using a microscale zebrafish epilepsy model to rapidly quantify in vivo antiseizure activity. The UHPLC-HRMS/MS profiling revealed the chemical diversity of the extracts and the presence of numerous bufadienolides, furostanols and ecdysteroids, also present in the decoction.

Zebrafish bioassay-guided isolation of antiseizure compounds from the Cameroonian medicinal plant Cyperus articulatus L.

BACKGROUND: Epilepsy is a chronic neurological disorder affecting more than 50 million people worldwide, of whom 80% live in low- and middle-income countries. Due to the limited availability of antiseizure drugs (ASDs) in these countries, medicinal plants are the first-line treatment for most epilepsy patients. In Cameroon, a decoction of Cyperus articulatus L. rhizomes is traditionally used to treat epilepsy. PURPOSE: The aim of this study was to identify and isolate the active compounds responsible for the antiseizure activity of C. articulatus in order to confirm both its traditional medicinal usage and previous in vivo studies on extracts of this plant in mouse epilepsy models. METHODS: The dried rhizomes of C. articulatus were extracted with solvents of increasing polaritie (hexane, dichloromethane, methanol and water). A traditional decoction and an essential oil were also prepared. These extracts were evaluated for antiseizure activity using a larval zebrafish seizure model with seizures induced by the GABAA antagonist pentylenetetrazole (PTZ). The hexane extract demonstrated the highest antiseizure activity and was therefore selected for bioassay-guided fractionation. The isolated bioactive compounds were characterized by classical spectroscopic methods. Since they were found to be volatile, they were quantified by GC-FID. In addition, the absorption of the active compounds through the gastrointestinal tract and the blood-brain barrier was evaluated using a hexadecane and a blood-brain barrier parallel artificial membrane permeability assays (HDM-PAMPA and PAMPA-BBB). RESULTS: The hexane extract of C. articulatus exhibited the highest antiseizure activity with a reduction of 93% of PTZ-induced seizures, and was therefore subjected to bioassay-guided fractionation in order to isolate the active principles. Four sesquiterpenoids were identified as cyperotundone (1), mustakone (2), 1,2-dehydro-α-cyperone (3) and sesquichamaenol (4) and exhibited significant antiseizure activity. These volatile compounds were quantified by GC in the hexane extract, the essential oil and the simulated traditional decoction. In addition, the constituents of the hexane extract including compounds 1 and 2 were found to cross the gastrointestinal barrier and the major compound 2 crossed the blood-brain barrier as well. CONCLUSION: These results highlight the antiseizure activity of various sesquiterpene compounds from a hexane extract of C. articulatus dried rhizomes and support its use as a traditional treatment for epilepsy.

Zebrafish-based identification of the antiseizure nucleoside inosine from the marine diatom Skeletonema marinoi.

With the goal of identifying neuroactive secondary metabolites from microalgae, a microscale in vivo zebrafish bioassay for antiseizure activity was used to evaluate bioactivities of the diatom Skeletonema marinoi, which was recently revealed as being a promising source of drug-like small molecules. A freeze-dried culture of S. marinoi was extracted by solvents with increasing polarities (hexane, dichloromethane, methanol and water) and these extracts were screened for anticonvulsant activity using a larval zebrafish epilepsy model with seizures induced by the GABAA antagonist pentylenetetrazole. The methanolic extract of S. marinoi exhibited significant anticonvulsant activity and was chosen for bioassay-guided fractionation, which associated the bioactivity with minor constituents. The key anticonvulsant constituent was identified as the nucleoside inosine, a well-known adenosine receptor agonist with previously reported antiseizure activities in mice and rat epilepsy models, but not reported to date as a bioactive constituent of microalgae. In addition, a UHPLC-HRMS metabolite profiling was used for dereplication of the other constituents of S. marinoi. Structures of the isolated compounds were elucidated by nuclear magnetic resonance and high-resolution spectrometry. These results highlight the potential of zebrafish-based screening and bioassay-guided fractionation to identify neuroactive marine natural products.

A below-ground herbivore shapes root defensive chemistry in natural plant populations.

Plants display extensive intraspecific variation in secondary metabolites. However, the selective forces shaping this diversity remain often unknown, especially below ground. Using Taraxacum officinale and its major native insect root herbivore Melolontha melolontha, we tested whether below-ground herbivores drive intraspecific variation in root secondary metabolites. We found that high M. melolontha infestation levels over recent decades are associated with high concentrations of major root latex secondary metabolites across 21 central European T. officinale field populations. By cultivating offspring of these populations, we show that both heritable variation and phenotypic plasticity contribute to the observed differences. Furthermore, we demonstrate that the production of the sesquiterpene lactone taraxinic acid ß-D-glucopyranosyl ester (TA-G) is costly in the absence, but beneficial in the presence of M. melolontha, resulting in divergent selection of TA-G. Our results highlight the role of soil-dwelling insects for the evolution of plant defences in nature.

A Latex Metabolite Benefits Plant Fitness under Root Herbivore Attack.

Plants produce large amounts of secondary metabolites in their shoots and roots and store them in specialized secretory structures. Although secondary metabolites and their secretory structures are commonly assumed to have a defensive function, evidence that they benefit plant fitness under herbivore attack is scarce, especially below ground. Here, we tested whether latex secondary metabolites produced by the common dandelion (Taraxacum officinale agg.) decrease the performance of its major native insect root herbivore, the larvae of the common cockchafer (Melolontha melolontha), and benefit plant vegetative and reproductive fitness under M. melolontha attack. Across 17 T. officinale genotypes screened by gas and liquid chromatography, latex concentrations of the sesquiterpene lactone taraxinic acid ß-D-glucopyranosyl ester (TA-G) were negatively associated with M. melolontha larval growth. Adding purified TA-G to artificial diet at ecologically relevant concentrations reduced larval feeding. Silencing the germacrene A synthase ToGAS1, an enzyme that was identified to catalyze the first committed step of TA-G biosynthesis, resulted in a 90% reduction of TA-G levels and a pronounced increase in M. melolontha feeding. Transgenic, TA-G-deficient lines were preferred by M. melolontha and suffered three times more root biomass reduction than control lines. In a common garden experiment involving over 2,000 T. officinale individuals belonging to 17 different genotypes, high TA-G concentrations were associated with the maintenance of high vegetative and reproductive fitness under M. melolontha attack. Taken together, our study demonstrates that a latex secondary metabolite benefits plants under herbivore attack, a result that provides a mechanistic framework for root herbivore driven natural selection and evolution of plant defenses below ground.