Fractionation and Extraction Optimization of Potentially Valuable Compounds and Their Profiling in Six Varieties of Two Nicotiana Species.

There is an increasingly urgent call to shift industrial processes from fossil fuel feedstock to sustainable bio-based resources. This change becomes of high importance considering new budget requirements for a carbon-neutral economy. Such a transformation can be driven by traditionally used plants that are able to produce large amounts of valuable biologically relevant secondary metabolites. Tobacco plants can play a leading role in providing value-added products in remote areas of the world. In this study, we propose a non-exhaustive list of compounds with potential economic interest that can be sourced from the tobacco plant. In order to optimize extraction methodologies, we first analyzed their physico-chemical properties using rapid solubility tests and high-resolution microfractionation techniques. Next, to identify an optimal extraction for a selected list of compounds, we compared 13 different extraction method-solvent combinations. We proceeded with profiling some of these compounds in a total of six varieties from Nicotiana tabacum and Nicotiana rustica species, identifying the optimal variety for each. The estimated expected yields for each of these compounds demonstrate that tobacco plants can be a superior source of valuable compounds with diverse applications beyond nicotine. Among the most interesting results, we found high variability of anatabine content between species and varieties, ranging from 287 to 1699 µg/g. In addition, we found that CGA (1305 µg/g) and rutin (7910 µg/g) content are orders of magnitude lower in the Burley variety as compared to all others.

BICS01 Mediates Reversible Anti-seizure Effects in Brain Slice Models of Epilepsy.

Drug-resistant epilepsy remains a significant clinical and societal burden, with one third of people with epilepsy continuing to experience seizures despite the availability of around 30 anti-seizure drugs (ASDs). Further, ASDs often have substantial adverse effects, including impacts on learning and memory. Therefore, it is important to develop new ASDs, which may be more potent or better tolerated. Here, we report the preliminary preclinical evaluation of BICS01, a synthetic product based on a natural compound, as a potential ASD. To model seizure-like activity in vitro, we prepared hippocampal slices from adult male Sprague Dawley rats, and elicited epileptiform bursting using high extracellular potassium. BICS01 (200 µM) rapidly and reversibly reduced the frequency of epileptiform bursting but did not change broad measures of network excitability or affect short-term synaptic facilitation. BICS01 was well tolerated following systemic injection at up to 1,000 mg/kg. However, we did not observe any protective effect of systemic BICS01 injection against acute seizures evoked by pentylenetetrazol. These results indicate that BICS01 is able to acutely reduce epileptiform activity in hippocampal networks. Further preclinical development studies to enhance pharmacokinetics and accumulation in the brain, as well as studies to understand the mechanism of action, are now required.

MicroRNAs in epilepsy: pathophysiology and clinical utility.

BACKGROUND: Temporal lobe epilepsy is a common and frequently intractable seizure disorder. Its pathogenesis is thought to involve large-scale alterations to the expression of genes controlling neurotransmitter signalling, ion channels, synaptic structure, neuronal death, gliosis, and inflammation. Identification of mechanisms coordinating gene networks in patients with temporal lobe epilepsy will help to identify novel therapeutic targets and biomarkers. MicroRNAs (miRNAs) are a family of small non-coding RNAs that control the expression levels of multiple proteins by decreasing mRNA stability and translation, and could therefore be key regulatory mechanisms and therapeutic targets in epilepsy. RECENT DEVELOPMENTS: In the past 5 years, studies have found changes in miRNA levels in the hippocampus of patients with temporal lobe epilepsy and in neural tissues from animal models of epilepsy. Early functional studies showed that silencing of brain-specific miR-134 using antisense oligonucleotides (antagomirs) had potent antiseizure effects in animal models, whereas genetic deletion of miR-128 produced fatal epilepsy in mice. Levels of certain miRNAs were also found to be altered in the blood of rodents after seizures. In the past 18 months, functional studies have identified nine novel miRNAs that appear to influence seizures or hippocampal pathology. Their targets include transcription factors, neurotransmitter signalling components, and modulators of neuroinflammation. New approaches to manipulate miRNAs have been tested, including injection of mimics (agomirs) to enhance brain levels of miRNAs. Altered miRNA expression has also been reported in other types of refractory epilepsy and our understanding of how miRNA levels are controlled has grown, with studies on DNA methylation indicating epigenetic regulation. Biofluids (blood) of patients with epilepsy have shown differences in quantity of circulating miRNAs, implying diagnostic biomarker potential. WHERE NEXT?: Recent functional studies need to be replicated to build a robust evidence base. The specific cell types in which miRNAs execute their functions and their primary targets have to be identified, to fully explain the phenotypic effects of modulating miRNAs. Delivery of large molecules such as antisense inhibitors or mimics to the brain poses a challenge, and the multi-targeting effects of miRNAs create additional risks of unanticipated side effects. Potential genetic variation in miRNAs should be explored as the basis for disease susceptibility. The latest findings provide a rich source of new miRNA targets, but substantial challenges remain before their role in the pathogenesis, diagnosis, and treatment of epilepsy can be translated into clinical practice.

Evaluation of the anti-inflammatory and antioxidative potential of four fern species from China intended for use as food supplements.

Inflammation plays a major role in many diseases, for instance in arteriosclerosis, rheumatoid arthritis, autoimmune disorders and cancer. Since many plants contain compounds with anti-inflammatory activity, their consumption may be able to prevent the development of inflammatory-based diseases. Edible ferns are some of the most important wild vegetables in China and have traditionally been used both for dietary and therapeutic purposes. In this study we investigated the anti-inflammatory and antioxidant potential of fern extracts from Matteuccia struthiopteris, Osmundajaponica, Matteuccia orientalis and Pteridium aquilinum intended for use as nutraceuticals. Two modes of action were investigated: the inhibition of the pro-inflammatory gene expression of interleukin-1 beta (IL1-ß) and interleukin-6 (IL6), and the gene expression of iNOS by LPS-elicited macrophages. The results showed a decrease of IL1-ß gene expression for the five fern extracts. This effect was more pronounced for the extracts prepared from the roots of O. japonica (IC50 of 17.8 µg/mL) and the young fronds of M orientalis (50.0 µg/mL). Regarding the indirect measurement of NO, via iNOS gene expression, an interesting decrease of 50% was obtained with the extract of M. orientalis fronds at a low concentration (20 µg/mL) compared with P. aquilinum fronds (160 µg/mL) and leaves of O. japonica. The latter showed a higher decrease but at a high concentration of extract (160 µg/mL). The five fern extracts were also evaluated for their ability to scavenge 2,2-diphenyl-l-picrylhydrazyl (DPPH) radicals and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). All fern extracts exhibited antioxidant effects but the roots of O. japonica and the fronds of M orientalis were most efficient. The HPLC-MS analysis of the constituents of the fern extracts confirmed the presence of chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, kaempferol and apigenin, molecules known to exhibit antiinflammatory and/or antioxidant properties.

The identification of naturally occurring neoruscogenin as a bioavailable, potent, and high-affinity agonist of the nuclear receptor RORα (NR1F1).

Plants represent a tremendous structural diversity of natural compounds that bind to many different human disease targets and are potentially useful as starting points for medicinal chemistry programs. This resource is, however, still underexploited due to technical difficulties with the identification of minute quantities of active ingredients in complex mixtures of structurally diverse compounds upon raw phytomass extraction. In this work, we describe the successful identification of a novel class of potent RAR-related orphan receptor alpha (RORα or nuclear receptor NR1F1) agonists from a library of 12,000 plant extract fractions by using an optimized, robust high-throughput cell-free screening method, as well as an innovative hit compound identification procedure through further extract deconvolution and subsequent structural elucidation of the active natural compound(s). In particular, we demonstrate that neoruscogenin, a member of the steroidal sapogenin family, is a potent and high-affinity RORα agonist, as shown by its activity in RORα reporter assays and from its effect on RORα target gene expression in vitro and in vivo. Neoruscogenin represents a universal pharmacological tool for RORα research due to its specific selectivity profile versus other nuclear receptors, its excellent microsomal stability, good bioavailability, and significant peripheral exposure in mouse.