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Alpha-tocotrienol (α-TCT) is a member of the vitamin E family. It has been reported to protect the brain against various pathologies including cerebral ischemia and neurodegeneration. However, it is still unclear if α-TCT exhibits beneficial effects during brain development. We hypothesized that treatment with α-TCT improves intracellular redox homeostasis supporting normal development of neurons. We found that primary hippocampal neurons isolated from rat feti grown in α-TCT-containing media achieved greater levels of neurite complexity compared to ethanol-treated control neurons. Neurons were treated with 1 μM α-TCT for 3 weeks, and media were replaced with fresh α-TCT every week. Treatment with α-TCT increased α-TCT levels (26 pmol/mg protein) in the cells, whereas the control neurons did not contain α-TCT. α-TCT-treated neurons produced adenosine triphosphate (ATP) at a higher rate and increased ATP retention at neurites, supporting formation of neurite branches. Although treatment with α-TCT alone did not change neuronal viability, neurons grown in α-TCT were more resistant to death at maturity. We further found that messenger RNA and protein levels of B-cell lymphoma-extra large (Bcl-xL) are increased by α-TCT treatment without inducing posttranslational cleavage of Bcl-xL. Bcl-xL is known to enhance mitochondrial energy production, which improves neuronal function including neurite outgrowth and neurotransmission. Therefore α-TCT-mediated Bcl-xL upregulation may be the central mechanism of neuroprotection seen in the α-TCT-treated group. In summary, treatment with α-TCT upregulates Bcl-xL and increases ATP levels at neurites. This correlates with increased neurite branching during development and with protection of mature neurons against oxidative stress.

Nature-derived bioactive compounds have emerged as promising candidates for the prevention and treatment of diverse chronic illnesses, including neurodegenerative diseases. However, the exact molecular mechanisms underlying their neuroprotective effects remain unclear. Most studies focus solely on the antioxidant activities of natural products which translate to poor outcome in clinical trials. Current therapies against neurodegeneration only provide symptomatic relief thereby underscoring the need for novel strategies to combat disease onset and progression. We have employed an environmental toxin-induced Drosophila Parkinson’s disease (PD) model as an inexpensive in vivo screening platform to explore neuroprotective potential of selected dietary flavonoids. We have identified a specific group of flavonoids known as flavones displaying protection against paraquat (PQ)-induced neurodegenerative phenotypes, involving reduced survival, mobility defects and enhanced oxidative stress. Interestingly, the other groups of investigated flavonoids, namely, the flavonones and flavonols failed to provide protection indicating a requirement of specific structural features that confer protection against PQ-mediated neurotoxicity in Drosophila. Based on our screen, the neuroprotective flavones lack a functional group substitution at the C3 and contain α,β-unsaturated carbonyl group. Furthermore, flavones-mediated neuroprotection is not solely dependent on antioxidant properties but also involves regulation of neuroinflammatory responses. Our data identify specific structural features of selected flavonoids that provide neuroprotection against environmental toxin-induced PD pathogenesis that can be explored for novel therapeutic interventions.Graphical Abstract

Understanding plant‐insect interactions is an active area of research in both ecology and evolution. Much attention has been focused on the impact of secondary metabolites in the host plant or fungi on these interactions. Plants and fungi contain a variety of biologically active compounds, and the secondary metabolite profile can vary significantly between individual samples. However, many experiments characterize the biological effects of only a single secondary metabolite or a subset of these compounds.Here, we develop an exhaustive extraction protocol using an accelerated solvent extraction protocol to recover the complete suite of cyclopeptides and other secondary metabolites found in Amanita phalloides (death cap mushrooms) and compare its efficacy to the “Classic” extraction method used in earlier works.We demonstrate that our extraction protocol recovers the full suite of cyclopeptides and other secondary metabolites in A. phalloides unlike the “Classic” method that favors polar cyclopeptides.Based on these findings, we provide recommendations for how to optimize protocols to ensure exhaustive extracts and also the best practices when using natural extracts in ecological experiments., While host plants and fungi contain a complex suite of metabolites, many studies examining plant‐insect interactions focus on the biological effect of a single compound. We developed a new protocol to exhaustively extract secondary metabolites from Amanita phalloides mushrooms. Based on our findings, we present recommendations of best practices for studies using secondary metabolite extracts.

Brain-derived neurotrophic factor (BDNF) and its receptor tyrosine receptor kinase B (TrkB) have been shown to play an important role in numerous neurological disorders, such as Alzheimer's disease. The identification of biologically active compounds interacting with TrkB serves as a drug discovery strategy to identify drug leads for neurological disorders. Here, we report effective immobilization of functional TrkB on magnetic iron oxide nanoclusters, where TrkB receptors behave as "smart baits" to bind compounds from mixtures and magnetic nanoclusters enable rapid isolation through magnetic separation. The presence of the immobilized TrkB was confirmed by specific antibody labeling. Subsequently, the activity of the TrkB on iron oxide nanoclusters was evaluated with ATP/ADP conversion experiments using a known TrkB agonist. The immobilized TrkB receptors can effectively identify binders from mixtures containing known binders, synthetic small molecule mixtures, and Gotu Kola (

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder with no cure. Despite intensive research, most of the currently available therapies are only effective in alleviating symptoms with no effect on disease progression. There is an urgent need for new therapeutics to impede disease progression. Natural products are valuable sources of bioactive compounds that can be exploited for novel therapeutic potential in PD pathogenesis. However, rapid screening of plant-derived natural products and characterization of bioactive compounds is costly and challenging. Drosophila melanogaster, commonly known as the fruit fly, has recently emerged as an excellent model for human neurodegenerative diseases, including PD. The high degree of conserved molecular pathways with mammalian models make Drosophila PD models an inexpensive solution to preliminary phases of target validation in the drug discovery pipeline. The present review provides an overview of drug discovery from natural extracts using Drosophila as a screening platform to evaluate the therapeutic potential of phytochemicals against PD.

The lack of suitable tools for the identification of potential drug leads from complex matrices is a bottleneck in drug discovery. Here, we report a novel method to screen complex matrices for new drug leads targeting transmembrane receptors. Using α3β4 nicotinic receptors as a model system, we successfully demonstrated the ability of this new tool for the specific identification and effective extraction of binding compounds from complex mixtures. The formation of cell-membrane coated nanoparticles was confirmed by transmission electron microscopy. In particular, we have developed a direct tool to evaluate the presence of functional α3β4 nicotinic receptors on the cell membrane. The specific ligand binding to α3β4 nicotinic receptors was examined through ligand fishing experiments and confirmed by high-performance liquid chromatography coupled with diode-array detection and electrospray ionization mass spectrometry. This tool has a great potential to transform the drug discovery process focusing on identification of compounds targeting transmembrane proteins, as more than 50% of all modern pharmaceuticals use membrane proteins as prime targets.

Parkinson’s disease (PD) is an age-associated neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons from the midbrain. Epidemiological studies have implicated exposures to environmental toxins like the herbicide, paraquat (PQ) as major contributors to PD etiology in both mammalian and invertebrate models. We have employed a PQ-induced PD model in Drosophila as an inexpensive in vivo platform to screen therapeutics from natural products. We have identified the polymethoxyflavonoid, GardeninA, with neuroprotective potential against PQ-induced parkinsonian symptoms involving reduced survival, mobility defects, and loss of dopaminergic neurons. GardeninA-mediated neuroprotection is not solely dependent on its antioxidant activities but also involves modulation of the neuroinflammatory and cellular death responses. Furthermore, we have successfully detected GardeninA bioavailability in the fly heads after oral administration using ultra-performance liquid chromatography and mass spectrometry. Our findings reveal a molecular mechanistic insight into GardeninA-mediated neuroprotection against environmental toxin-induced PD pathogenesis for novel therapeutic intervention.

Amyotrophic lateral sclerosis (ALS) is caused by selective and progressive loss of spinal, bulbar and cortical motoneurons and leads to irreversible paralysis, loss of speech, inability to swallow and respiratory malfunctions with the eventual death of the affected individual in a rapid disease course. Several suggested molecular pathways are reviewed including SOD1 gene mutation, protein nitrosylation, phosphorylation and oxidative stress, excitotoxicity, glutamate transporter deprivation, mitochondrial involvement, protein aggregation and motor neuron trophic factors. The role of insulin and its receptor in the brain is described. It is very possible that in 90% of the sporadic ALS cases, the cause of the motor neuron degeneration is different or that multiple mechanisms are involved that would need drugs with multiple mechanisms or action. Several marketed drugs have been selected for clinical trials. Only two drugs have been approved by the FDA as showing positive effect in ALS: Riluzole and Edaravone. Two other drugs that have a significant benefit in ALS are Talampanel and Tamoxifen. The results for modulation of the neurotrophic factor Insulin Growth Factor-1 (IGF1) as a potential treatment are inconclusive. Several compounds are discussed that show a positive effect in the mouse model but which have failed in clinical trials. New approaches using different modalities such as peptides, proteins and stem cells are promising. Our ability to design better drugs would be enhanced by investigating the endogenous factors in neuron death, protein aggregation and oxidative stress that would improve our understanding of the potential pathways that result in neurodegeneration.

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder with no cure. Despite intensive research, most of the currently available therapies are only effective in alleviating symptoms with no effect on disease progression. There is an urgent need for new therapeutics to impede disease progression. Natural products are valuable sources of bioactive compounds that can be exploited for novel therapeutic potential in PD pathogenesis. However, rapid screening of plant-derived natural products and characterization of bioactive compounds is costly and challenging. Drosophila melanogaster, commonly known as the fruit fly, has recently emerged as an excellent model for human neurodegenerative diseases, including PD. The high degree of conserved molecular pathways with mammalian models make Drosophila PD models an inexpensive solution to preliminary phases of target validation in the drug discovery pipeline. The present review provides an overview of drug discovery from natural extracts using Drosophila as a screening platform to evaluate the therapeutic potential of phytochemicals against PD.

The treatment of epilepsy remains difficult mostly since almost 30% of patients suffer from pharmacoresistant forms of the disease. Therefore, there is an urgent need to search for new antiepileptic drug candidates. Previously, it has been shown that 4-alkyl-5-substituted-1,2,4-triazole-3-thione derivativatives possessed strong anticonvulsant activity in a maximal electroshock-induced seizure model of epilepsy. In this work, we examined the effect of the chemical structure of the 1,2,4-triazole-3-thione-based molecules on the anticonvulsant activity and the binding to voltage-gated sodium channels (VGSCs) and GABAA receptors. Docking simulations allowed us to determine the mode of interactions between the investigated compounds and binding cavity of the human VGSC. Selected compounds were also investigated in a panel of ADME-Tox assays, including parallel artificial membrane permeability assay (PAMPA), single cell gel electrophoresis (SCGE) and cytotoxicity evaluation in HepG2 cells. The obtained results indicated that unbranched alkyl chains, from butyl to hexyl, attached to 1,2,4-triazole core are essential both for good anticonvulsant activity and strong interactions with VGSCs. The combined in-vivo, in-vitro and in-silico studies emphasize 4-alkyl-5-substituted-1,2,4-triazole-3-thiones as promising agents in the development of new anticonvulsants.

This research aims to investigate synergistic and antagonistic antioxidant interactions for binary mixtures of common monoterpenes. The activity was determined spectrophotometrically with the use of the DPPH• assay. Significant antioxidant synergism, between two different monoterpenes, was observed for a mixture of inactive p-cymene and one of the compounds, characterized by moderate antioxidant activity, e.g. (±)-citronellal, α-phellandrene or α-terpinene. It has been proven that the activity of (±)-citronellal is increased in the presence of hydrocarbons possessing the aromatic nucleophilic ring, which may react with electrophiles. A possible reaction mechanism was proposed for the synergistic antioxidant effect of p-cymene and (±)-citronellal. This research opens new perspectives for studying interactions between isoprene derivatives, found in essential oils. Copyright © 2016 John Wiley & Sons, Ltd.

Mass spectrometry has developed into an important tool that compliments ultraviolet and diode array detector detection techniques. Mass spectrometry has become a sort of universal technique enabling detection of different classes of compounds but also the determination of the molecular mass and isotope composition of compounds. The use of tandem mass spectrometry delivers more data required for an unambiguous identification of the analyzed compounds. In tandem mass spectrometry, the selected ions formed in the ionization source are preselected in the first stage of analysis and subsequently fragmented. Product ions, formed in the second step, are separated and detected afterward. For those focusing on untargeted analysis of samples and dealing with the detection of unknown compounds with similar structures and physicochemical properties, mass spectrometry has only become really useful with the advent of high-resolution mass spectrometry.

Secondary plant metabolites are evolutionary-designed molecules that interact with multiple biological targets in human organisms. Identification of pharmacologically active phytochemicals is usually a time consuming and costly process. Cellular membrane affinity chromatography (CMAC) allows the detection of secondary metabolites present in complex natural matrices, e.g. plant extracts and their interactions with the immobilized fully-functional transmembrane proteins. After the isolation process of the binding compounds, CMAC columns can be used to study the binding process between the potential new ligands and the immobilized transmembrane protein target. The following parameters can be determined using CMAC columns: binding affinity (Kd), association rate constant (kon), dissociation rate constant (koff) and the equilibrium constant for complex formation (K). This review summarizes the preparation steps and the use of CMAC columns in the drug discovery process of new potential drug leads present in complex natural matrices.

The research aims to indicate which of the structural elements of monoterpenes are responsible for their antioxidant activity. The activity was determined spectrophotometrically with the use of the DPPH(•) assay. It has been shown that π bonds are responsible for the chain-breaking antioxidant activity of monoterpenes. It has been proved, for the first time, that blocking of conjugated double bonds leads to a decrease of the antioxidant activity of monoterpenes. A probable reaction mechanism between monoterpenes and DPPH(•) has been proposed. It has been indicated that the antioxidant activity of monoterpenes strongly depends on the polarity of solvent used in the experiments. The presented results may stimulate additional research in the field of terpenoid antioxidants.

The α3β4α5 nAChR has been recently shown to be a useful target for smoking cessation pharmacotherapies. Herein, we report on the development and characterization of the α3β4α5 nicotinic receptor column by frontal displacement chromatography. The binding affinity of the nicotine and minor alkaloids found in tobacco smoke condensates were determined for both the α3β4 and α3β4α5 nicotinic receptors. It was demonstrated that while no subtype selectivity was observed for nicotine and nornicotine, anabasine was selective for the α3β4α5 nicotinic receptor. The non-competitive inhibitor binding site was also studied and it was demonstrated while mecamylamine was not selective between subtypes, buproprion showed subtype selectivity for the α3β4 nicotinic receptor. The application of this methodology to complex mixtures was then carried out by screening aqueous-alcoholic solutions of targeted plant extracts, including Lycopodium clavatum L. (Lycopodiaceae) and Trigonella foenum graecum L. (Fabaceae) against both the α3β4 and α3β4α5 nAChRs.

Fourteen phenolic compounds (flavonoids and phenolic acids) were isolated and 19 were identified in the aerial parts of Triticum aestivum L. The structures of these compounds were established on the basis of the data obtained by electrospray ionization-mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR) techniques. T. aestivum L. was found to be rich in flavones, especially in luteolin derivatives. Three of the isolated compounds, including luteolin 6-C-[6Glc″-O-E-caffeoyl-β-D-glucopyranosyl(1″→2)-β-glucopyranoside], luteolin 6-C-[5Rib″-O-E-feruoyl-β-D-ribofuranosyl(1″→2)-β-glucopyranoside], and 3',4',5'-O-trimethyltricetin 7-O-[β-D-glucuropyranosyl(1″→2)-β-D-glucopyranoside], have been reported for the first time in the plant kingdom. The amount of individual phenolics, in winter wheat, was also determined. Additionally, the free radical scavenging potential of the isolated compounds was tested in a simple and rapid thin-layer chromatography-2,2-diphenyl-1-picrylhydrazyl radical test (TLC-DPPH•) with image processing.

For the first time Trifolium scabrum L. was researched for its phenolic profile. Sixteen phenolics (isoflavones and flavonoids) were isolated and identified in the aerial parts of T. scabrum L. Their structures were established by electrospray ionization-mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR) techniques. Quantitative analysis of individual phenolics performed by means of ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) was based on calibration curves obtained for previously isolated standard compounds. Additionally, the free radical scavenging potential of these substances was assessed by means of a simple benchtop thin-layer chromatography-2,2-diphenyl-1-picrylhydrazyl radical (TLC-DPPH(•)) bioassay. Thus, T. scabrum L. can be regarded as a potential source of estrogenic and antioxidant compounds, both of significance in the pharmaceutical as well as the food industry. The results show that T. scabrum L. can be considered as a natural and very good commercial source of phenolic compounds (mainly isoflavones).

HPTLC is one of the most widely applied methods in phytochemical analysis. It is due to its numerous advantages, e.g., it is the only chromatographic method offering the option of presenting the results as an image. Other advantages include simplicity, low costs, parallel analysis of samples, high sample capacity, rapidly obtained results, and possibility of multiple detection. HPTLC provides identification as well as quantitative results. It also enables the identification of adulterants. In case of complex samples, the resolving power of traditional one-dimensional chromatography is usually inadequate, hence special modes of development are required. Multidimensional and multimodal HPTLC techniques include those realized in one direction (UMD, IMD, GMD, BMD, AMD) as well as typical two-dimensional methods realized on mono- or bi-layers. In this manuscript, an overview on variable multidimensional and multimodal methods, applied in the analysis of phytochemical samples, is presented.