Investigation on the reactivity of isoxazol-5-ones towards 1,2-diaza-1,3-dienes: new entry to variously substituted (imidazol-2-yl)acetate and 1,3-oxazin-6-one derivatives

1,2-Diaza-1,3-dienes (DDs) undergo, under neutral conditions, N-nucleophilic attack from a 4-ethoxycarbonylisoxazol-5-one derivative. The first aza-Michael addition is followed by an intramolecular second, affording a fused heterobicyclic system that, upon ring opening and decarboxylation processes, gives rise to novel substituted imidazoles with an acetate functionality in the 2-position. On the contrary, under the same reaction conditions, 3-phenylisoxazol-5-one provides a double Michael addition at two units of DD involving first the C-4 and then the N-2 of the heterocycle. The resulting diadduct spontaneously undergoes ring-opening/ring-closing process that concludes with a ring enlargement of the heterocycle providing the 1,3-oxazin-6-one derivative.     

A Study on the Stability and Enzymatic Activity of Yeast Alcohol Dehydrogenase in Presence of the Self-Assembling Block Copolymer Poloxamer 407

Yeast alcohol dehydrogenase (ADH) is an enzyme widely studied for biotechnological applications due to its involvement in fermentation industry, and various attempts to improve its catalytic properties and its thermal stability have been carried out. In this paper, the influence of a block copolymer (Poloxamer 407) on ADH enzymatic activity and thermal behaviour has been studied in order to get new insights about the use of poloxamers in formulation of sustained release systems for therapeutic proteins. Poloxamer 407 has the ability to form micelles and gel due to its self-assembling and thermoresponsive properties. The effect of the copolymer towards thermal stress and pH changes, which often reduce enzymes activity it has been investigated by means of enzymatic assays and differential scanning calorimetry. Results showed that at pH?9.1 and 7.3, the Poloxamer in the form of unimeric, micellar and gel state is able to effectively preserve the enzyme from thermoinactivation. In addition by calorimetric data Poloxamer 407 has showed an effect in preserving ADH from aggregation at pH?7.3. In conclusion, Poloxamer 407 seems to be very effective in protecting ADH from stress related events, like alkaline inactivation and aggregation.     

Two‐Photon Excitation of a Plasmonic Nanoswitch Monitored by Single‐Molecule Fluorescence Microscopy

Visible‐light excitation of the surface plasmon band of silver nanoplates can effectively localize and concentrate the incident electromagnetic field enhancing the photochemical performance of organic molecules. Herein, the first single‐molecule study of the plasmon‐assisted isomerization of a photochrome‐fluorophore dyad, designed to switch between a nonfluorescent and a fluorescent state in response to the photochromic transformation, is reported. The photochemistry of the switchable assembly, consisting of a photochromic benzooxazine chemically conjugated to a coumarin moiety, is examined in real time with total internal reflection fluorescence microscopy in the presence of silver nanoplates excited with a 633 nm laser. The metallic nanostructures significantly enhance the visible light‐induced performance of the photoconversion, which normally requires ultraviolet excitation. The resulting ring‐open isomer is strongly fluorescent and can also be excited at 633 nm. These stochastic emission events are used to monitor photochromic activation and show quadratic dependence on incident power. The utilization of a single laser wavelength for both photochromic activation and excitation effectively mimics a pseudo two‐colours system.     

Cannabinoid Receptor Modulation of Neurogenesis: ST14A Striatal Neural Progenitor Cells as a Simplified In Vitro Model

The endocannabinoid system (ECS) is involved in the modulation of several basic biological processes, having widespread roles in neurodevelopment, neuromodulation, immune response, energy homeostasis and reproduction. In the adult central nervous system (CNS) the ECS mainly modulates neurotransmitter release, however, a substantial body of evidence has revealed a central role in regulating neurogenesis in developing and adult CNS, also under pathological conditions. Due to the complexity of investigating ECS functions in neural progenitors in vivo, we tested the suitability of the ST14A striatal neural progenitor cell line as a simplified in vitro model to dissect the role and the mechanisms of ECS-regulated neurogenesis, as well as to perform ECS-targeted pharmacological approaches. We report that ST14A cells express various ECS components, supporting the presence of an active ECS. While CB1 and CB2 receptor blockade did not affect ST14A cell number, exogenous administration of the endocannabinoid 2-AG and the synthetic CB2 agonist JWH133 increased ST14A cell proliferation. Phospholipase C (PLC), but not PI3K pharmacological blockade negatively modulated CB2-induced ST14A cell proliferation, suggesting that a PLC pathway is involved in the steps downstream to CB2 activation. On the basis of our results, we propose ST14A neural progenitor cells as a useful in vitro model for studying ECS modulation of neurogenesis, also in prospective in vivo pharmacological studies.     

Biotransformation of Cortisone with Rhodococcus rhodnii: Synthesis of New Steroids

Cortisone is a steroid widely used as an anti-inflammatory drug able to suppress the immune system, thus reducing inflammation and attendant pain and swelling at the site of an injury. Due to its numerous side effects, especially in prolonged and high-dose therapies, the development of the pharmaceutical industry is currently aimed at finding new compounds with similar activities but with minor or no side effects. Biotransformations are an important methodology towards more sustainable industrial processes, according to the principles of “green chemistry”. In this work, the biotransformation of cortisone with Rhodococcus rhodnii DSM 43960 to give two new steroids, i.e., 1,9β,17,21-tetrahydoxy-4-methyl-19-nor-9β-pregna-1,3,5(10)-trien-11,20-dione and 1,9β,17,20β,21-pentahydoxy-4-methyl-19-nor-9β-pregna-1,3,5(10)-trien-11-one, is reported. These new steroids have been fully characterized.     

Chemical Composition,In Vitro Bioaccessibility and Antioxidant Activity of Polyphenolic Compounds from Nutraceutical Fennel Waste Extract

Fennel (Foeniculum vulgare Mill.) waste contains a broad range of bioactive molecules, including polyphenols, which have poor bioaccessibility during gastrointestinal digestion. This work aimed to investigate the bioaccessibility of total phenolic compounds and the antioxidant capacity during simulated gastrointestinal digestion using two nutraceutical formulations based on non-acid-resistant (NAR) and acid-resistant (AR) capsules containing aqueous-based extracts from fennel waste. Moreover, to obtain a comprehensive investigation of the polyphenolic constituents of the fennel waste extract, a high-resolution mass spectrometry (Q-Orbitrap) analysis was performed. Notably, chlorogenic acids, such as 4-caffeoylquinic acid and 3,4-dicaffeoylquinic acid, were the most detected compounds found in assayed samples (1.949 and 0.490 mg/g, respectively). After in vitro gastrointestinal digestion, the extract contained in AR capsules displayed higher bioaccessibility in both the duodenal and colonic stages (1.96 and 5.19 mg GAE/g, respectively) than NAR capsules (1.72 and 3.50 mg GAE/g, respectively), suggesting that the acidic gastric conditions negatively affected the polyphenol compounds released from the NAR capsules. Therefore, the aqueous extract of fennel waste could be proposed as an innovative and easily available source of dietary polyphenols. Furthermore, the use of an AR capsule could improve the polyphenol bioaccessibility and can be proposed as a nutraceutical formulation.     

Delivery of Thyronamines (TAMs) to the Brain: A Preliminary Study

Recent reports highlighted the significant neuroprotective effects of thyronamines (TAMs), a class of endogenous thyroid hormone derivatives. In particular, 3-iodothyronamine (T1AM) has been shown to play a pleiotropic role in neurodegeneration by modulating energy metabolism and neurological functions in mice. However, the pharmacological response to T1AM might be influenced by tissue metabolism, which is known to convert T1AM into its catabolite 3-iodothyroacetic acid (TA1). Currently, several research groups are investigating the pharmacological effects of T1AM systemic administration in the search of novel therapeutic approaches for the treatment of interlinked pathologies, such as metabolic and neurodegenerative diseases (NDDs). A critical aspect in the development of new drugs for NDDs is to know their distribution in the brain, which is fundamentally related to their ability to cross the blood–brain barrier (BBB). To this end, in the present study we used the immortalized mouse brain endothelial cell line bEnd.3 to develop an in vitro model of BBB and evaluate T1AM and TA1 permeability. Both drugs, administered at 1 µM dose, were assayed by high-performance liquid chromatography coupled to mass spectrometry. Our results indicate that T1AM is able to efficiently cross the BBB, whereas TA1 is almost completely devoid of this property.     

Evaluation of Antioxidant and Enzyme Inhibition Properties of Croton hirtus L’Hér. Extracts Obtained with Different Solvents

Croton hirtus L’Hér methanol extract was studied by NMR and two different LC-DAD-MSⁿ using electrospray (ESI) and atmospheric pressure chemical ionization (APCI) sources to obtain a quali-quantitative fingerprint. Forty different phytochemicals were identified, and twenty of them were quantified, whereas the main constituents were dihydro α ionol-O-[arabinosil(1-6) glucoside] (133 mg/g), dihydro β ionol-O-[arabinosil(1-6) glucoside] (80 mg/g), β-sitosterol (49 mg/g), and isorhamnetin-3-O-rutinoside (26 mg/g). C. hirtus was extracted with different solvents—namely, water, methanol, dichloromethane, and ethyl acetate—and the extracts were assayed using different in vitro tests. The methanolic extracts presented the highest 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS), and ferric reducing antioxidant power (FRAP) values. All the tested extracts exhibited inhibitory effects on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), with a higher activity observed for dichloromethane (AChE: 5.03 and BChE: 16.41 mgGALAE/g), while the methanolic extract showed highest impact against tyrosinase (49.83 mgKAE/g). Taken together, these findings suggest C. hirtus as a novel source of bioactive phytochemicals with potential for commercial development.     

NMR Profiling of Ononis diffusa Identifies Cytotoxic Compounds against Cetuximab-Resistant Colon Cancer Cell Lines

In the search of new natural products to be explored as possible anticancer drugs, two plant species, namely Ononis diffusa and Ononis variegata, were screened against colorectal cancer cell lines. The cytotoxic activity of the crude extracts was tested on a panel of colon cancer cell models including cetuximab-sensitive (Caco-2, GEO, SW48), intrinsic (HT-29 and HCT-116), and acquired (GEO-CR, SW48-CR) cetuximab-resistant cell lines. Ononis diffusa showed remarkable cytotoxic activity, especially on the cetuximab-resistant cell lines. The active extract composition was determined by NMR analysis. Given its complexity, a partial purification was then carried out. The fractions obtained were again tested for their biological activity and their metabolite content was determined by 1D and 2D NMR analysis. The study led to the identification of a fraction enriched in oxylipins that showed a 92% growth inhibition of the HT-29 cell line at a concentration of 50 µg/mL.