Effect of Hydroxytyrosol Derivatives of Donepezil on the Activity of Enzymes Involved in Neurodegenerative Diseases and Oxidative Damage.

Monoamine oxidase and xanthine oxidase inhibitors represent useful multi-target drugs for the prevention, attenuation, and treatment of oxidative damage and neurodegenerative disorders. Chimeric molecules, constituted by naturally derived compounds linked to drugs, represent lead compounds to be explored for the discovery of new synthetic drugs acting as enzyme inhibitors. We have previously reported that seven hydroxytyrosol-donepezil hybrid compounds play a protective role in an in vitro neuronal cell model of Alzheimer's disease. In this work, we analyzed the effects exerted by the hybrid compounds on the activity of monoamine oxidase A (MAO-A) and B (MAO-B), as well as on xanthine oxidase (XO), enzymes involved in both neurodegenerative disorders and oxidative stress. The results pointed to the identification, among the compounds tested, of selective inhibitors between the two classes of enzymes. While the 4-hydroxy-3-methoxyphenethyl 1-benzylpiperidine-4-carboxylate- (HT3) and the 4-hydroxyphenethyl 1-benzylpiperidine-4-carboxylate- donepezil derivatives (HT4) represented the best inhibitors of MAO-A, with a scarce effect on MAO-B, they were almost ineffective on XO. On the other hand, the 4,5-dihydroxy-2-nitrophenethyl 1-benzylpiperidine-4-carboxylate donepezil derivative (HT2), the least efficient MAO inhibitor, acted like the best XO inhibitor. Therefore, the differential enzymatic targets identified among the hybrid compounds synthesized enhance the possible applications of these polyphenol-donepezil hybrids in neurodegenerative disorders and oxidative stress.

Storage Effects on Bioactive Phenols in Calabrian Monovarietal Extra Virgin Olive Oils Based on the EFSA Health Claim.

The beneficial properties of extra virgin olive oil (EVOO) on lipids blood levels were recognized by the European Food Safety Authority (EFSA) with a health claim, specifically referring to EVOOs containing at least 5 mg of hydroxytyrosol and its secoiridoids derivatives per 20 g of oil. The main purpose of the work was to characterize the phenolic profile of two commercially available Calabrian monovarietal EVOOs (Nocellara del Belice, VN; Dolce di Rossano, VDR), and to study the effect of one-year storage on secoiridoids composition, by monthly controls. A new UHPLC-ESI-HRMS method was developed and validated, thus facilitating the EFSA claim application and allowing producers to valorize their products. Seven biologically active compounds were chosen: tyrosol, hydroxytyrosol, oleocanthal, oleacein, oleuropein aglycone, verbascoside, and oleuropein. LODs and LOQs were 0.001-0.02 mg g-1 and 0.002-0.08 mg g-1, respectively. The variation coefficients were ≤20% and the percentage of recovery was between 89-109%. During the 12-month storage period, the concentration of selected compounds ranged between 1258.78-1478.91 mg Kg-1 for VN, and 1408.22-2071.45 mg Kg-1 for VDR, with a decrease of 15% and 32% respectively. The method allows an accurate quantification of EVOO phenols thus being useful to certify the nutraceutical properties of olive oil.

Hydroxytyrosol-Donepezil Hybrids Play a Protective Role in an In Vitro Induced Alzheimer's Disease Model and in Neuronal Differentiated Human SH-SY5Y Neuroblastoma Cells.

Alzheimer's disease (AD) is the most common neurodegenerative pathology among progressive dementias, and it is characterized by the accumulation in the brain of extracellular aggregates of beta-amyloid proteins and neurofibrillary intracellular tangles consisting of τ-hyperphosphorylated proteins. Under normal conditions, beta-amyloid peptides exert important trophic and antioxidant roles, while their massive presence leads to a cascade of events culminating in the onset of AD. The fibrils of beta-amyloid proteins are formed by the process of fibrillogenesis that, starting from individual monomers of beta-amyloid, can generate polymers of this protein, constituting the hypothesis of the "amyloid cascade". To date, due to the lack of pharmacological treatment for AD without toxic side effects, chemical research is directed towards the realization of hybrid compounds that can act as an adjuvant in the treatment of this neurodegenerative pathology. The hybrid compounds used in this work include moieties of a hydroxytyrosol, a nitrohydroxytyrosol, a tyrosol, and a homovanillyl alcohol bound to the N-benzylpiperidine moiety of donepezil, the main drug used in AD. Previous experiments have shown different properties of these hybrids, including low toxicity and antioxidant and chelating activities. The purpose of this work was to test the effects of hybrid compounds mixed with Aß1-40 to induce fibrillogenesis and mimic AD pathogenesis. This condition has been studied both in test tubes and by an in vitro model of neuronal differentiated human SH-SY5Y neuroblastoma cells. The results obtained from test tube experiments showed that some hybrids inhibit the activity of the enzymes AChE, BuChE, and BACE-1. Cell experiments suggested that hybrids could inhibit fibrillogenesis, negatively modulating caspase-3. They were also shown to exert antioxidant effects, and the acetylated hybrids were found to be more functional and efficient than nonacetylated forms.

Functional Compounds of Cold-Pressed Pomegranate Seed Oil: Fatty Acids and Phytosterols Profile as Quality Biomarkers for Origin Discrimination.

Cold-pressed pomegranate seed oil (PSO) is a product of the extraction of non-edible pomegranate seeds. Its unique chemical composition in terms of both polyunsaturated fatty acids, especially punicic acid (PA), and secondary metabolites, such as phytosterols, tocopherols and phenols, make it an interesting functional ingredient for food enrichment. It is not clear if the biomarkers profile of PSO depends to factors connected to the geographical origin of seeds. This work presents a statistical comparative analysis, concerning biomolecules composition and geographical origin of 32 commercial cold-pressed PSOs, performed by principal component analysis. The study discriminates between Turkish and Italian PSOs, on the base of the fatty acid profile and phytosterols, and not on the tocopherols and phenols. These results confirmed PA as the main characteristic biomarker of oil genuineness and, for the first time, disclosed a statistically relevant variability of phytosterols, which can be proposed as quality biomarkers for discrimination of geographical origins.

The Double-Edged Sword of Oleuropein in Ovarian Cancer Cells: From Antioxidant Functions to Cytotoxic Effects.

Oleuropein plays a key role as a pro-oxidant as well as an antioxidant in cancer. In this study, the activity of oleuropein, in an in vitro model of ovarian (OCCs) and breast cancer cells (BCCs) was investigated. Cell viability and cell death were analyzed. Oxidative stress was measured by CM-H2DCFDA flow cytometry assay. Mitochondrial dysfunction was evaluated based on mitochondrial reactive oxygen species (ROS) and GPX4 protein levels. Further, the effects on iron metabolism were analyzed by measuring the intracellular labile iron pool (LIP). We confirmed that high doses of oleuropein show anti-proliferative and pro-apoptotic activity on HEY and MCF-7 cells. Moreover, our results indicate that low doses of oleuropein impair cell viability without affecting the mortality of cells, and also decrease the LIP and ROS levels, keeping them unchanged in MCF-7 cells. For the first time, our data show that low doses of oleuropein reduce erastin-mediated cell death. Interestingly, oleuropein decreases the levels of intracellular ROS and LIP in OCCs treated with erastin. Noteworthily, we observed an increased amount of ROS scavenging enzyme GPX4 together with a consistent reduction in mitochondrial ROS, confirming a reduction in oxidative stress in this model.

Catalytic amination of lactic acid using Ru-zeolites.

In this work we investigate the synthesis of alanine from lactic acid, a biobased platform chemical, using ammonia as a nitrogen source and Ru/zeolite catalysts. We report a high alanine selectivity when using Ru/BEA of 80-93%. Reaction side products were identified as ethanol, propionic acid or propanamide and the reaction mechanism was investigated. We further optimised reaction conditions resulting in turn over numbers five times higher than previously reported and could reduce Ru leaching by 30-40%. However, leaching and catalyst stability remains a concern. Furthermore, we critically analyse the benefits of Ru/zeolites versus their stability under the basic, high temperature reaction conditions.

The Highly Efficient Synthesis of 1,2-Disubstituted Benzimidazoles Using Microwave Irradiation.

The benzimidazole ring of the heterocyclic pharmacophores is one of the most widespread and studied systems in nature. The benzimidazole derivative synthesis study is a crucial point for the development of a clinically available benzimidazole-based drug. Here, we report a simple microwave assisted method for the synthesis of 1,2-disubstituted benzimidazoles. The combination of the molar ratio of N-phenyl-o-phenylenediamine:benzaldehyde (1:1) using microwave irradiation and only 1% mol of Er(OTf)3 provides an efficient and environmental mild access to a diversity of benzimidazoles under solvent-free conditions. The proposed method allows for the obtainment of the desired products in a short time and with very high selectivity.

Oleil Hydroxytyrosol (HTOL) Exerts Anti-Myeloma Activity by Antagonizing Key Survival Pathways in Malignant Plasma Cells.

Polyphenols from olive oil are endowed with several biological activities. Chemical modifications have been recently applied to these compounds to improve their therapeutic activity in different pathological settings, including cancer. Herein, we describe the in vitro effects on multiple myeloma (MM) cells of oleil hydroxytyrosol (HTOL), a synthetic fatty ester of natural hydroxytyrosol with oleic acid. HTOL reduced the viability of various human MM cell lines (HMCLs), even when co-cultured with bone marrow stromal cells, triggering ER stress, UPR and apoptosis, while it was not cytotoxic against healthy peripheral blood mononuclear cells or B lymphocytes. Whole-transcriptome profiling of HTOL-treated MM cells, coupled with protein expression analyses, indicate that HTOL antagonizes key survival pathways for malignant plasma cells, including the undruggable IRF4-c-MYC oncogenic axis. Accordingly, c-MYC gain- and loss-of-function strategies demonstrate that HTOL anti-tumor activity was, at least in part, due to c-MYC targeting. Taken together, these findings underscore the anti-MM potential of HTOL, providing the molecular framework for further investigation of HTOL-based treatments as novel anti-cancer agents.

Novel Hydroxytyrosol-Donepezil Hybrids as Potential Antioxidant and Neuroprotective Agents.

It is well-accepted that the endogenous antioxidant protection system progressively decays in elderly people, and that the oxidative stress contributes to different neurodegenerative disorders such as Alzheimer's Diseases (AD). The lower incidence of AD in countries which feature the Mediterranean Diet was associated to the high consumption of extra virgin olive oil and its polyphenolic fraction, in particular hydroxytyrosol. The protective role of these bio-phenols against oxidative stress, suggested that we combine their antioxidant/free radical scavenging activity with donepezil, an active ingredient which has just been approved for the treatment of AD. Different synthetic strategies were tested to conjugate the two different synthons in good yields. Additionally, a nitro-hydroxytyrosol derivative was synthesized to extend the application to other neurodegeneration inflammatory models. Then, their bioactivity was measured in different chemical and biological tests on a human neuroblastoma cell line (SHSY-5Y). Remarkable results on cell viability and the regulation of the redox state of cells were obtained. All hybrids showed negligible cell death under 1 µM and are stable and non toxic. Reactive oxygen species (ROS) measurements showed that the nitro-hybrid was the more effective one at reducing the ROS amount to physiological values. Then, in light of the bio-metal hypothesis of diverse neurodegenerative disorders, we tested these new compounds on the chelation properties of redox-active metals. The nitro-hybrid was able to chelate all of the tested metal cations, suggesting that we propose it as potential lead compound for a new class of neuroprotective antioxidant agents.

Quali-quantitative monitoring of chemocatalytic cellulose conversion into lactic acid by FT-NIR spectroscopy.

Chemocatalytic conversion of cellulose into lactic acid is a valuable alternative to simple sugar fermentation. Nevertheless, the procedures still need optimization to be translated to the industrial scale. Such translation would benefit by on-line monitoring of reaction parameters by fast, inexpensive, direct spectroscopic techniques. In this work, we propose the application of FT-NIR spectroscopy as a suitable analytical tool for monitoring the chemocatalytic conversion of cellulose into lactic acid. Comparison between different FT-NIR spectra at different reaction temperatures and times was exploited to qualitatively indicate the feasibility of the reaction. Besides, an FT-NIR prediction model was proposed for rapidly estimating the molar distribution of cellulose catalytic degradation components in the reaction mixtures. The calibration model was based on reference samples analysed by HPLC. The model was validated by an external validation set. Relevant statistical values of Ratio Performance to Deviations (RPD) referred to both calibration and external validation were obtained, thus demonstrating the potential of such analytical technique in process monitoring.

Semi-synthesis as a tool for broadening the health applications of bioactive olive secoiridoids: a critical review.

Covering: 2005 up to 2020Olive bioactive secoiridoids are recognized as natural antioxidants with multiple beneficial effects on human health. Nevertheless, the study of their biological activity has also disclosed some critical aspects associated with their application. Firstly, only a few of them can be extracted in large amounts from their natural matrix, namely olive leaves, drupes, oil and olive mill wastewater. Secondly, their application as preventive agents and drugs is limited by their low membrane permeability. Thirdly, the study of their biological fate after administration is complicated by the absence of pure analytical standards. Accordingly, efficient synthetic methods to obtain natural and non-natural bioactive phenol derivatives have been developed. Among them, semi-synthetic protocols represent efficient and economical alternatives to total synthesis, combining efficient extraction protocols with efficient catalytic conversions to achieve reasonable amounts of active molecules. The aim of this review is to summarize the semi-synthetic protocols published in the last fifteen years, covering 2005 up to 2020, which can produce natural olive bioactive phenols scarcely available by extractive procedures, and new biophenol derivatives with enhanced biological activity. Moreover, the semi-synthetic protocols to produce olive bioactive phenol derivatives as analytical standards are also discussed. A critical analysis of the advantages offered by semi-synthesis compared to classical extraction methods or total synthesis protocols is also performed.

In Vitro Anthelminthic Efficacy of Aqueous Pomegranate (Punica granatum L.) Extracts against Gastrointestinal Nematodes of Sheep.

The worldwide increased difficulty to counteract gastrointestinal nematode (GIN) infection in sheep, due to progressing anthelmintic resistance, has led to the evaluation of other alternative helminth control options, mainly from plants. The anthelmintic efficacy of an aqueous Punica granatum macerate was evaluated in sheep naturally infected by GIN in southern Italy. The macerate was chemically characterized by chromatographic analysis coupled with high-resolution mass spectrometry (LC/HRMS) and an aliquot was concentrated to obtain a dry extract. A part was characterized, the remaining washed with methanol to obtain an insoluble residue and methanol phase. In the methanol fraction, the quantitatively predominant gallic acid was purified to obtain the pure molecule. The three fractions thus obtained were used for in vitro studies (i.e., egg hatch test) to verify anthelmintic efficacy. For this purpose, fecal samples were collected from sheep naturally infected by GINs. Fractions were diluted in H2O/DMSO 0.5% at 1.00, 0.5, 0.25, 0.125, 0.05, and 0.005 mg/mL concentrations. Thiabendazole (0.25 and 0.5 mg/mL) and deionized water were used as positive and negative controls, respectively. Egg hatch test results indicated that all fractions caused a significant (p < 0.05) egg hatch inhibition within 48 h of exposure highlighting a high (>82%) efficacy in vitro at all tested doses. Maximal egg hatching inhibition effect was exhibited by the methanol fraction (99.3% and 89.3% at 1 and 0.005 mg/mL concentrations), followed by the insoluble residue and gallic acid (94.7% and 85.3% and 94.0% and 82.7% at 1 and 0.005 mg/mL, respectively). The current study validated the anthelmintic potential of traditional P. granatum macerate against GIN infection in sheep, thus highlighting the role of gallic acid as principal component and justifying a need to undertake further in vivo studies on these ethno-veterinary remedies.

Combined Ultrasound/Microwave Chemocatalytic Method for Selective Conversion of Cellulose into Lactic Acid.

Cellulose is the main component of lignocellulosic biomass. Its direct chemocatalytic conversion into lactic acid (LA), a powerful biobased chemical platform, represents an important, and more easily scalable alternative to the fermentative way. In this paper, we present the selective hydrothermal conversion of cellulose and simple sugars into LA, under mild reaction conditions in presence of ErCl3 grafted on the mesoporous silica (MCM-41) surface. High yields and selectivity were obtained for the conversion of sugars under microwave (MW) irradiation at a relatively low temperature (200 °C) and short reaction times (10 min) under microwave (MW) irradiation. Ultrasounds (US) pre-treatment was investigated to reduce the cellulose crystallinity, before the MW-assisted conversion, providing LA with a yield of 64% within 90 min at 220 °C below the subcritical water conditions with increased operational safety. We finally discuss the scalability of the process and the recyclability of the catalyst.

Green Synthesis of Privileged Benzimidazole Scaffolds Using Active Deep Eutectic Solvent.

The exploitation and use of alternative synthetic methods, in the face of classical procedures that do not conform to the ethics of green chemistry, represent an ever-present problem in the pharmaceutical industry. The procedures for the synthesis of benzimidazoles have become a focus in synthetic organic chemistry, as they are building blocks of strong interest for the development of compounds with pharmacological activity. Various benzimidazole derivatives exhibit important activities such as antimicrobial, antiviral, anti-inflammatory, and analgesic activities, and some of the already synthesized compounds have found very strong applications in medicine praxis. Here we report a selective and sustainable method for the synthesis of 1,2-disubstituted or 2-substituted benzimidazoles, starting from o-phenylenediamine in the presence of different aldehydes. The use of deep eutectic solvent (DES), both as reaction medium and reagent without any external solvent, provides advantages in terms of yields as well as in the work up procedure of the reaction.

Anti-tumor Activity and Epigenetic Impact of the Polyphenol Oleacein in Multiple Myeloma.

Olive oil contains different biologically active polyphenols, among which oleacein, the most abundant secoiridoid, has recently emerged for its beneficial properties in various disease contexts. By using in vitro models of human multiple myeloma (MM), we here investigated the anti-tumor potential of oleacein and the underlying bio-molecular sequelae. Within a low micromolar range, oleacein reduced the viability of MM primary samples and cell lines even in the presence of bone marrow stromal cells (BMSCs), while sparing healthy peripheral blood mononuclear cells. We also demonstrated that oleacein inhibited MM cell clonogenicity, prompted cell cycle blockade and triggered apoptosis. We evaluated the epigenetic impact of oleacein on MM cells, and observed dose-dependent accumulation of both acetylated histones and α-tubulin, along with down-regulation of several class I/II histone deacetylases (HDACs) both at the mRNA and protein level, providing evidence of the HDAC inhibitory activity of this compound; conversely, no effect on global DNA methylation was found. Mechanistically, HDACs inhibition by oleacein was associated with down-regulation of Sp1, the major transactivator of HDACs promoter, via Caspase 8 activation. Of potential translational significance, oleacein synergistically enhanced the in vitro anti-MM activity of the proteasome inhibitor carfilzomib. Altogether, these results indicate that oleacein is endowed with HDAC inhibitory properties, which associate with significant anti-MM activity both as single agent or in combination with carfilzomib. These findings may pave the way to novel potential anti-MM epi-therapeutic approaches based on natural agents.

Synthesis and preliminary evaluation of the anti-cancer activity on A549 lung cancer cells of a series of unsaturated disulfides.

We synthesized a series of small symmetrical unsaturated disulfides by a multi-step reaction starting from organic alcohols, and we performed a preliminary test to evaluate the effect of these compounds on the viability of A549 lung cancer cells. The garlic-derived natural compound diallyl disulfide, known for its anticancer activity, was used as the lead compound in this study. We synthesized five DADS analogues having different carbon chain lengths and different positions of the double bonds. Two analogues exhibited a promising antitumor activity in vitro, and the allylic double bond did not seem to be the main driving force.

Hydrolases-mediated transformation of oleuropein into demethyloleuropein.

Phenolic compounds present in extra virgin olive oil have recently attracted considerable attention due to their pharmacological activities. Among them oleacein (3,4-DHPEA-EDA), structurally related to oleochantal (4-HPEA-EDA), is one of the most studied. 3,4-DHPEA-EDA has been synthesized through decarboxylation of demethyloleuropein catalyzed by Er(OTf)3. Demethyloleuropein is extracted from black olives drupes in very limited amounts and only in particular periods of the year. The availability of demethyloleuropein could be increased by a selective hydrolysis of the methyl ester moiety of oleuropein, a secoiridoid present in large amount in olive leaves. In this work we describe a new enzymatic method for carrying out a selective hydrolysis of oleuropein via the screening of a panel of hydrolases (lipases, esterases and proteases). Among all the enzymes tested the best results was obtained using α-chymotrypsyn from bovine pancreas as biocatalyst, thus revealing a classic example of catalytic enzyme promiscuity.

Qualitative and quantitative analysis of the proautophagic activity of Citrus flavonoids from Bergamot Polyphenol Fraction.

Bergamot Polyphenol Fraction (BPF®) is a natural mixture of Citrus flavonoids extracted from processed bergamot fruits. It has been shown to counteract cardiovascular risk factors and to prevent liver steatosis in rats and patients. Hepatic effects of BPF correlate with its ability to stimulate liver autophagy. Six aglyconic flavonoids have been identified in the proautophagic fraction of the hydrolysis product of BPF (A-BPF): naringenin, hesperetin, eridictyol, diosmetin, apigenin and luteolin. We report here the output parameters of high resolution mass spectrometry analysis of these flavonoids and chemical structures of their parent compounds. The second set of data shows the proautophagic activity of BPF flavonoids in a hepatic cell line HepG2 analyzed by a flow cytometry approach. The method is based on the red to green fluorescence intensity ratio analysis of DsRed -LC3- GFP, which is stably expressed in HepG2 cells. Proportional analysis of ATG indexes allowed us to address a relative contribution of individual compounds to the proautophagic activity of the A-BPF mixture and evaluate if the effect was additive. Qualitative analysis of ATG indexes compared the effects of flavonoids at equal concentrations in the presence and absence of palmitic acid and chloroquine. The Excel files reporting the analysis of flow cytometry data are available in the public repository.

Biorenewable Deep Eutectic Solvent for Selective and Scalable Conversion of Furfural into Cyclopentenone Derivatives.

The development of novel synthetic routes to produce bioactive compounds starting from renewable sources has become an important research area in organic and medicinal chemistry. Here, we present a low-cost procedure for the tunable and selective conversion of biomass-produced furfural to cyclopentenone derivatives using a mixture of choline chloride and urea as a biorenewable deep eutectic solvent (DES). The proposed medium is a nontoxic, biodegradable, and could be reused up to four times without any unfavorable effect on the reaction yield. The process is tunable, clean, cheap, simple and scalable and meets most of the criteria; therefore, it can be considered as an environmental sustainable process in a natural reaction medium.