Development, Optimization, and Comparison of Different Sample Pre-Treatments for Simultaneous Determination of Vitamin E and Vitamin K in Vegetables

The absence of vitamin E from the diet can lead to cardiovascular disease, cancer, cataracts, and premature aging. Vitamin K deficiency can lead to bleeding disorders. These fat-soluble vitamins are important nutritional factors that can be determined in different methods in vegetables. In this work, the simultaneous determination of α-tocopherol, α-tocopheryl acetate, phylloquinone, and menaquinone-4 by gas chromatography–mass spectrometry (GC–MS) has been optimized using both direct injection and solid phase microextraction (SPME). Three different sample pre-treatment approaches based on: (A) solid–liquid–liquid–liquid extraction (SLE–LLE), (B) SLE, and (C) SPME were then applied to extract the target analytes from vegetables samples using menaquinone as internal standard. All the procedures allowed the determination of the target analytes in onion, carrot, celery, and curly kale samples. Similar results were obtained with the three different approaches, even if the one based on SPME offers the best performance, together with a reduced use of solvent, time consumption, and experimental complexity, which makes it the preferable option for industrial applications

Extra Virgin Olive Oil Extracts Modulate the Inflammatory Ability of Murine Dendritic Cells Based on Their Polyphenols Pattern: Correlation between Chemical Composition and Biological Function

Extra virgin olive oil (EVOO) represents one of the most important health-promoting foods whose antioxidant and anti-inflammatory activities are mainly associated to its polyphenols content. To date, studies exploring the effect of EVOO polyphenols on dendritic cells (DCs), acting as a crosstalk between the innate and the adaptive immune response, are scanty. Therefore, we studied the ability of three EVOO extracts (cv. Coratina, Cima di Mola/Coratina, and Casaliva), characterized by different polyphenols amount, to regulate DCs maturation in resting conditions or after an inflammatory stimulus. Cima di Mola/Coratina and Casaliva extracts were demonstrated to be the most effective in modulating DCs toward an anti-inflammatory profile by reduction of TNF and IL-6 secretion and CD86 expression, along with a down-modulation of Il-1β and iNOS expression. From factorial analysis results, 9 polyphenols were tentatively established to play a synergistic role in modulating DCs inflammatory ability, thus reducing the risk of chronic inflammation

Effects of Sweet Cherry Polyphenols on Enhanced Osteoclastogenesis Associated With Childhood Obesity

Childhood obesity is associated with the development of severe comorbidities, such as diabetes, cardiovascular diseases, and increased risk of osteopenia/osteoporosis and fractures. The status of low-grade inflammation associated to obesity can be reversed through an enhanced physical activity and by consumption of food enrich of anti-inflammatory compounds, such as omega-3 fatty acids and polyphenols. The aim of this study was to deepen the mechanisms of bone impairment in obese children and adolescents through the evaluation of the osteoclastogenic potential of peripheral blood mononuclear cells (PBMCs), and the assessment of the serum levels of RANKL and osteoprotegerin (OPG). Furthermore, we aimed to evaluate the in vitro effects of polyphenol cherry extracts on osteoclastogenesis, as possible dietary treatment to improve bone health in obese subjects. High RANKL levels were measured in obese with respect to controls (115.48 ± 35.20 pg/ml vs. 87.18 ± 17.82 pg/ml; p < 0.01), while OPG levels were significantly reduced in obese than controls (378.02 ± 61.15 pg/ml vs. 436.75 ± 95.53 pg/ml, respectively, p < 0.01). Lower Ad-SoS- and BTT Z-scores were measured in obese compared to controls (p < 0.05). A significant elevated number of multinucleated TRAP+ osteoclasts (OCs) were observed in the un-stimulated cultures of obese subjects compared to the controls. Interestingly, obese subjects displayed a higher percentage of CD14+/CD16+ than controls. Furthermore, in the mRNA extracts of obese subjects we detected a 2.5- and 2-fold increase of TNFα and RANKL transcripts compared to controls, respectively. Each extract of sweet cherries determined a dose-dependent reduction in the formation of multinucleated TRAP+ OCs. Consistently, 24 h treatment of obese PBMCs with sweet cherry extracts from the three cultivars resulted in a significant reduction of the expression of TNFα. In conclusion, the bone impairment in obese children and adolescents is sustained by a spontaneous osteoclastogenesis that can be inhibited in vitro by the polyphenol content of sweet cherries. Thus, our study opens future perspectives for the use of sweet cherry extracts, appropriately formulated as nutraceutical food, as preventive in healthy children and therapeutic in obese ones

Dry powder formulations from olive oil wastewater with high nutraceutical properties

Objectives The olive oil extraction process leads to a high amount of olive oil waste water (OW), a rich source of polyphenolic compounds that can exert a wide range of beneficial effects on human health, most of them due to polyphenolic compounds antioxidant properties [1, 2]. In order to minimize the impact to the environment and to valorize the sustainable development of several local products, a concentrated solution from OW was obtained and, starting from this solution, we developed a stable dry powder formulation which maintains its high nutraceutical properties. Methods On the concentrated olive oil wastewater, here called HP30, a preformulation study was performed in order to select the appropriate excipients in terms of solubility, viscosity and dispersibility of the systems. In particular, we tested four different excipients, here called E1 (polysaccharide), E2 (polysaccharide), E3 (oligosaccharide) and E4 (desiccant) and after the above mentioned studies we selected the three giving better results, i.e. E1 E3 and E4. Different amounts of the selected excipient were used to obtain the formulations from HP30, called F1 (5% E1, 10% E3), F3 (5% E1, 30% E3), F1b (5% E1, 10% E3, 2% E4) and F3b (5% E1, 30% E3, 2% E4), respectively. DSC studies were carried out to evaluate the thermal properties of the prepared formulations and to optimize the freeze-drying process. All obtained formulations were characterized by the Folin assay to analyze the polyphenols content with respect to the HP30 and by stability studies. Results The HP30, a concentrated solution from the OW was treated to obtain stable solid formulations with high nutraceutical properties. After preliminary studies we selected three excipients and we prepared four different formulation called F1, F3, F1b and F3b. All formulations were obtained by an optimized freeze-drying process. From a macroscopic point of view all powders showed a light brown colour, they had good flow properties with respect to the sticking HP30 powder and they resulted stable up to five months storage under normal conditions of humidity and temperature. The Folin assay, carried out on the formulations, did not show significant differences in term of polyphenolic amount with respect to the HP30 for the formulations F1 and F1b, while a reduction of 20 % w/w was evidenced on F3 and F3b powders. So, we can assume that the freeze-dryied process is an useful method to obtain solid formulations from the OW not significantly modifying the composition of the principal components and that F1 and F1b are promising nutraceutical product obtained from olive oil wastewater. References: [1] Casamenti F. & Stefani M. “Olive polyphenols: new promising agents to combat aging-associated neurodegeneration” Expert Review of Neurotherapeutics, 2016 [2] Cornwell D, “Nutritional benefit of olive oil: the biological effects of hydroxytyrosol and its arylatingquinone adducts” Journal of agricultural and food chemistry, 200

Lubeluzole Repositioning as Chemosensitizing Agent on Multidrug-Resistant Human Ovarian A2780/DX3 Cancer Cells

In a previous paper, we demonstrated the synergistic action of the anti-ischemic lubeluzole (Lube S) on the cytotoxic activity of doxorubicin (Dox) and paclitaxel in human ovarian cancer A2780 and lung cancer A549 cells. In the present paper, we extended in vitro the study to the multi-drug-resistant A2780/DX3 cell line to verify the hypothesis that the Dox and Lube S drug association may potentiate the antitumor activity of this anticancer compound also in the context of drug resistance. We also evaluated some possible mechanisms underlying this activity. We analyzed the antiproliferative activity in different cancer cell lines. Furthermore, apoptosis, Dox accumulation, MDR1 downregulation, ROS, and NO production in A2780/DX3 cells were also evaluated. Our results confirm that Lube S improves Dox antiproliferative and apoptotic activities through different mechanisms of action, all of which may contribute to the final antitumor effect. Moderate stereoselectivity was found, with Lube S significantly more effective than its enantiomer (Lube R) and the corresponding racemate (Lube S/R). Docking simulation studies on the ABCB1 Cryo-EM structure supported the hypothesis that Lube S forms a stable MDR1-Dox-Lube S complex, which hampers the protein transmembrane domain flipping and blocks the efflux of Dox from resistant A2780/DX3 cells. In conclusion, our in vitro studies reinforce our previous hypothesis for repositioning the anti-ischemic Lube S as a potentiating agent in anticancer chemotherapy

Dry powder formulations from olive oil wastewater with high nutraceutical properties

Objectives The olive oil extraction process leads to a high amount of olive oil waste water (OW), a rich source of polyphenolic compounds that can exert a wide range of beneficial effects on human health, most of them due to polyphenolic compounds antioxidant properties [1, 2]. In order to minimize the impact to the environment and to valorize the sustainable development of several local products, a concentrated solution from OW was obtained and, starting from this solution, we developed a stable dry powder formulation which maintains its high nutraceutical properties. Methods On the concentrated olive oil wastewater, here called HP30, a preformulation study was performed in order to select the appropriate excipients in terms of solubility, viscosity and dispersibility of the systems. In particular, we tested four different excipients, here called E1 (polysaccharide), E2 (polysaccharide), E3 (oligosaccharide) and E4 (desiccant) and after the above mentioned studies we selected the three giving better results, i.e. E1 E3 and E4. Different amounts of the selected excipient were used to obtain the formulations from HP30, called F1 (5% E1, 10% E3), F3 (5% E1, 30% E3), F1b (5% E1, 10% E3, 2% E4) and F3b (5% E1, 30% E3, 2% E4), respectively. DSC studies were carried out to evaluate the thermal properties of the prepared formulations and to optimize the freeze-drying process. All obtained formulations were characterized by the Folin assay to analyze the polyphenols content with respect to the HP30 and by stability studies. Results The HP30, a concentrated solution from the OW was treated to obtain stable solid formulations with high nutraceutical properties. After preliminary studies we selected three excipients and we prepared four different formulation called F1, F3, F1b and F3b. All formulations were obtained by an optimized freeze-drying process. From a macroscopic point of view all powders showed a light brown colour, they had good flow properties with respect to the sticking HP30 powder and they resulted stable up to five months storage under normal conditions of humidity and temperature. The Folin assay, carried out on the formulations, did not show significant differences in term of polyphenolic amount with respect to the HP30 for the formulations F1 and F1b, while a reduction of 20 % w/w was evidenced on F3 and F3b powders. So, we can assume that the freeze-dryied process is an useful method to obtain solid formulations from the OW not significantly modifying the composition of the principal components and that F1 and F1b are promising nutraceutical product obtained from olive oil wastewater. References: [1] Casamenti F. & Stefani M. “Olive polyphenols: new promising agents to combat aging-associated neurodegeneration” Expert Review of Neurotherapeutics, 2016 [2] Cornwell D, “Nutritional benefit of olive oil: the biological effects of hydroxytyrosol and its arylatingquinone adducts” Journal of agricultural and food chemistry, 200

Optimization of Microwave-Assisted Extraction of Antioxidants from Bamboo Shoots of Phyllostachys pubescens

Bamboo is a well-known medicinal plant in Southeast Asia that recently has attracted attention for its high polyphenol content and its medical and nutraceutical applications. In this work, polyphenols have been recovered for the first time by microwave-assisted extraction (MAE) from an unusual Italian cultivar of Phyllostachys pubescens bamboo shoots. The eects of three independent variables, such as extraction time, temperature, and solid/liquid ratio, on polyphenol recovery yield were investigated and successfully optimized through the response surface methodology. We demonstrated that MAE is an excellent polyphenols extraction technique from bamboo shoots because the total phenolic content obtained under microwave irradiation optimal conditions (4 min at 105 C with 6.25 mg/mL ratio) was about eight-fold higher than that obtained with the conventional extraction method. Furthermore, higher total flavonoid content was also obtained under MAE. Consistent with these results, MAE enhanced the extract antioxidant properties with significant improved DPPH, ABTS, and FRAP scavenging ability. Therefore, this innovative extraction process enhances the recovery of biologically active compounds from Phyllostachys pubescens bamboo shoots with a dramatic reduction of time and energy consumption, which paves the way for its industrial application in functional food production

hERG stereoselective modulation by mexiletine-derived ureas: Molecular docking study, synthesis, and biological evaluation.

Long QT syndrome (LQTS) is a disorder of cardiac electrophysiology resulting in life-threatening arrhythmias; nowadays, only a few drugs are available for the management of LQTS. Focusing our attention on LQT2, one of the most common subtypes of LQTS caused by mutations in the human ether-à-go-go-related gene (hERG), in the present work, the stereoselectivity of the recently discovered mexiletine-derived urea 8 was investigated on the hERG potassium channel. According to preliminary in silico predictions, in vitro studies revealed a stereoselective behavior, with the meso form showing the greatest hERG opening activity. In addition, functional studies on guinea pig isolated left atria, aorta, and ileum demonstrated that 8 does not present any cardiac or intestinal liability in our ex vivo studies. Due to its overall profile, (R,S)-8 paves the way for the design and development of a new series of compounds potentially useful in the treatment of both congenital and drug-induced forms of LQTS.Fondazione Telethon—Italy, Grant/Award Number: # GGP19134S

Lubeluzole: from anti-ischemic drug to preclinical antidiarrheal studies.

BACKGROUND: Lubeluzole, a neuroprotective anti-ischemic drug, was tested for its ability to act as both antibiotic chemosensitizing and antipropulsive agent for the treatment of infectious diarrhea. METHODS: In the present report, the effect of lubeluzole against antidiarrheal target was tested. The antimicrobial activity towards Gram-positive and Gram-negative bacteria was investigated together with its ability to affect ileum and colon contractility. RESULTS: Concerning the antimicrobial activity, lubeluzole showed synergistic effects when used in combination with minocycline against four common Gram-positive and Gram-negative bacteria (Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853, and Escherichia coli ATCC 25922), although relatively high doses of lubeluzole were required. In ex vivo experiments on sections of gut smooth muscles, lubeluzole reduced the intestinal contractility in a dose-dependent manner, with greater effects observed on colon than on ileum, and being more potent than reference compounds otilonium bromide and loperamide. CONCLUSION: All above results identify lubeluzole as a possible starting compound for the development of a novel class of antibacterial adjuvants endowed with spasmolytic activity

Synthesis and Evaluation of Voltage-Gated Sodium Channel Blocking Pyrroline Derivatives Endowed with Both Antiarrhythmic and Antioxidant Activities

Under the hypothesis that cardioprotective agents might benefit from synergism between antiarrhythmic activity and antioxidant properties, a small series of mexiletine analogues were coupled with the 2,2,5,5-tetramethylpyrroline moiety, known for its antioxidant effect, in order to obtain dual-acting drugs potentially useful in the protection of the heart against post-ischemic reperfusion injury. The pyrroline derivatives reported herein were found to be more potent as antiarrhythmic agents than mexiletine and displayed antioxidant activity. The most interesting tetramethylpyrroline congener, a tert-butyl-substituted analogue, was at least 100 times more active as an antiarrhythmic than mexiletine