Targeting Proteolysis with Cyanogenic Glycoside Amygdalin Induces Apoptosis in Breast Cancer Cells.

BACKGROUND: Breast cancer is the most diagnosed cancer among women, and its incidence and mortality are rapidly growing worldwide. In this regard, plant-derived natural compounds have been shown to be effective as chemotherapeutic and preventative agents. Apricot kernels are a rich source of nutrients including proteins, lipids, fibers, and phenolic compounds and contain the aromatic cyanogenic glycoside amygdalin that has been shown to exert a cytotoxic effect on cancer cells by affecting the cell cycle, inducing apoptosis, and regulating the immune function. METHODS: Here, we describe a previously unexplored proapoptotic mechanism of action of amygdalin in breast cancer (MCF7) cells that involves the modulation of intracellular proteolysis. For comparative purposes, the same investigations were also conducted upon cell treatment with two apricot kernel aqueous extracts from Prunus armeniaca L. RESULTS: We observed that both the 20S and 26S proteasome activities were downregulated in the MCF7 cells upon 24 h treatments. Simultaneously, the autophagy cascade resulted in being impaired due to cathepsin B and L inhibition that also contributed to a reduction in cancer cell migration. The inhibition of these proteolytic systems finally promoted the activation of apoptotic events in the MCF7 cells. CONCLUSION: Collectively, our data unveil a novel mechanism of the anticancer activity of amygdalin, prompting further investigations for potential application in cancer preventative strategies.

Fiber-Sample Distance, An Important Parameter To Be Considered in Headspace Solid-Phase Microextraction Applications.

To define and control the parameters which impact headspace solid-phase microextraction (HS-SPME), it is important to reach the highest level of reproducibility. The present study aims to assess, for the first time, the effect of fiber-sample distance during HS-SPME in pre-equilibrium conditions. Analyses were primarily performed on mixtures of standard volatiles compounds (alkanes, alcohols, organic acids) designed in our lab and then on various food matrices (wine, chicken, cheese, tea), repeating already published experiments. Extractions were performed varying fiber penetration depths (10-60 mm) at different times (10-60 min) and temperatures of extraction (30-80 °C). The study revealed that variation of the distance between the fiber and the sample into the vial clearly impacts the results obtained during HS-SPME when conditions are such that no equilibrium is reached in HS. For example, in wine analysis, the percentage of octanoic acid at 80 °C was higher at 40 mm (7.5 ± 0.2%) than that at 20 mm (4.4 ± 0.3%). Moreover, regardless of the extraction temperature, the lower the time of extraction, the stronger the dependence on the fiber-sample distance. Indeed, at 60 °C, the obtained response factors for octadecane at 20 and 40 mm of fiber penetration were 21.8 and 44.5, respectively, after 10 min of extraction, 54.1 and 71.0 after 30 min, and 79.4 and 82.4 after 60 min of extraction. The analyses have been here corroborated by a theoretical model based on the diffusion equation. Therefore, to improve the method robustness during HS-SPME studies, we suggest specifying the fiber penetration depth or the fiber-sample distance with the other parameters of extraction.

Compensate for or Minimize Matrix Effects? Strategies for Overcoming Matrix Effects in Liquid Chromatography-Mass Spectrometry Technique: A Tutorial Review.

In recent decades, mass spectrometry techniques, particularly when combined with separation methods such as high-performance liquid chromatography, have become increasingly important in pharmaceutical, bio-analytical, environmental, and food science applications because they afford high selectivity and sensitivity. However, mass spectrometry has limitations due to the matrix effects (ME), which can be particularly marked in complex mixes, when the analyte co-elutes together with other molecules, altering analysis results quantitatively. This may be detrimental during method validation, negatively affecting reproducibility, linearity, selectivity, accuracy, and sensitivity. Starting from literature and own experience, this review intends to provide a simple guideline for selecting the best operative conditions to overcome matrix effects in LC-MS techniques, to obtain the best result in the shortest time. The proposed methodology can be of benefit in different sectors, such as pharmaceutical, bio-analytical, environmental, and food sciences. Depending on the required sensitivity, analysts may minimize or compensate for ME. When sensitivity is crucial, analysis must try to minimize ME by adjusting MS parameters, chromatographic conditions, or optimizing clean-up. On the contrary, to compensate for ME analysts should have recourse to calibration approaches depending on the availability of blank matrix. When blank matrices are available, calibration can occur through isotope labeled internal standards and matrix matched calibration standards; conversely, when blank matrices are not available, calibration can be performed through isotope labeled internal standards, background subtraction, or surrogate matrices. In any case, an adjusting of MS parameters, chromatographic conditions, or a clean-up are necessary.

The influence of different types of preparation (espresso and brew) on coffee aroma and main bioactive constituents.

Coffee is one of the most popular hot drinks in the world; it may be prepared by several methods, but the most common forms are boiled (brew) and pressurized (espresso). Analytical studies on the substances responsible for the pleasant aroma of roasted coffee have been carried out for more than 100 years. Brew coffee and espresso coffee (EC) have a different and peculiar aroma profile, demonstrating the importance of the brewing process on the final product sensorial quality. Concerning bioactive compounds, the extraction mechanism plays a crucial role. The differences in the composition of coffee brew in chlorogenic acids and caffeine content is the result of the different procedures of coffee preparation. The aim of the present review is to detail how the brewing process affects coffee aroma and composition.

Quantification of caffeine, trigonelline and nicotinic acid in espresso coffee: the influence of espresso machines and coffee cultivars.

Caffeine, trigonelline and nicotinic acid are important bioactive constituents of coffee. In this work, the combination of different water temperatures and pressures in the settings of the espresso coffee (EC) machine was evaluated, to assess how these factors influence how effectively caffeine, trigonelline and nicotinic acid are extracted from both Arabica and Robusta samples. The proposed analytical method, based on a high performance liquid chromatography (HPLC) system coupled to a variable wavelength detector (VWD), showed good linearity (R²> 0.9985) and good recoveries (71-92%); after validation for three monitored compounds, the method was used to analyze 20 commercial samples. The combination of a temperature of 92 °C and pressure at 7 or 9 bar seems to be the ideal setting for the most efficient extraction of these compounds and consequently for their intake; the compound extracted in the greatest quantity was caffeine, which was in the range of 116.87-199.68 mg in a 25 ml cup of coffee.

Anionic polysaccharides for stabilization and sustained release of antimicrobial peptides.

Chemical and enzymatic in vivo degradation of antimicrobial peptides represents a major challenge for their therapeutic use to treat bacterial infections. In this work, anionic polysaccharides were investigated for their ability to increase the chemical stability and achieve sustained release of such peptides. The investigated formulations comprised a combination of antimicrobial peptides (vancomycin (VAN) and daptomycin (DAP)) and anionic polysaccharides (xanthan gum (XA), hyaluronic acid (HA), propylene glycol alginate (PGA) and alginic acid (ALG)). VAN dissolved in buffer of pH 7.4 and incubated at 37 °C showed first order degradation kinetics with a reaction rate constant kobs of 5.5 × 10-2 day-1 corresponding with a half-life of 13.9 days. However, once VAN was present in a XA, HA or PGA-based hydrogel, kobs decreased to (2.1-2.3) × 10-2 day-1 while kobs was not affected in an alginate hydrogel and a dextran solution (5.4 × 10-2 and 4.4 × 10-2 day-1). Under the same conditions, XA and PGA also effectively decreased kobs for DAP (5.6 × 10-2 day-1), whereas ALG had no effect and HA even increased the degradation rate. These results demonstrate that the investigated polysaccharides (except ALG for both peptides and HA for DAP) slowed down the degradation of VAN and DAP. DSC analysis was used to investigate on polysaccharide ability to bind water molecules. Rheological analysis highlighted that the polysaccharides containing VAN displayed an increase in G' of their formulations, pointing that the peptides interaction act as crosslinker of the polymer chains. The obtained results suggest that the mechanism of stabilization of VAN and DAP against hydrolytic degradation is conferred by electrostatic interactions between the ionizable amine groups of the drugs and the anionic carboxylate groups of the polysaccharides. This, in turn, results in a close proximity of the drugs to the polysaccharide chain, where the water molecules have a lower mobility and, therefore, a lower thermodynamic activity.

A novel treatment and derivatization for quantification of residual aromatic diisocyanates in polyamide resins.

In the scientific context, the environmental and healthy impact of polymers is more related to the residual monomer content rather than their macromolecular structure, due to the monomer capability to interact with membrane cells. For this a novel method to stabilize and quantify residual monomeric isocyanates in high thermal resistance polyamide resins (PAs) has been developed. This new analytical method resulted in an improvement concerning the quantification of residual aromatic diisocyanates in viscous polymeric matrices by using a simple and cheap technique like HPLC-VWD. Diisocyanate monomers were derivatized with dibutylamine, resulting in stable urea derivatives that were simultaneously analysed and quantified. The method was applied to solvent-based polyamide resins, used as primary electrical insulation, for avoiding additional step of solvent removing before the analysis. The quantification of residual monomers answers to the provisions imposed by European Regulation N. 1907/2006 (REACH) for polymer registration, and the necessity of an early evaluation of the occupational risk associated with the use of diisocyanates, due to their toxicity and high reactivity towards moisture.

Chemical, Antioxidant, and Antimicrobial Properties of the Peel and Male Flower By-Products of Four Varieties of Punica granatum L. Cultivated in the Marche Region for Their Use in Cosmetic Products.

We are now seeing an increase in the production of agri-food waste, which is an essential resource for the recovery of bioactive compounds that may be employed as innovative natural ingredients in cosmetics. To date, the approach to cosmetics preservation has seen a significant shift in the search for biological components that give healthier alternatives for customers and help businesses operate in an environmentally friendly manner. To achieve this goal, we studied pomegranate extracts using the peel and, for the first time, extracts from the male flowers of a wide pomegranate variety cultivated in the Marche region, specifically, the Wonderful, Mollar de Elche, Parfianka, and less-studied G1 varieties. We studied the phenol compounds profile, antioxidant capacity, antimicrobial activity, and cell viability of the obtained pomegranate extracts. The identification and quantification of phenol compounds belonging to different classes, such as hydrolysable tannins, hydroxybenzoic acid, hydroxycinnamic acid, dihydroflavonol, gallocatechin, and anthocyanins, were performed using UPLC-ESI-MS/MS. Punicalagin isomers and punicalin resulted in the most abundant polyphenols found in the peel and male flower extracts. Mollar de Elche 2020 peel extract revealed a high concentration of punicalagin A and B (7206.4 mg/kg and 5812.9), while the content of gallic acid revealed high results in the G1 and Parfianka varieties. All extracts were spectrophotometrically analysed to determine their total phenol content (TPC) using the Folin-Ciocalteu method and their antioxidant capacity (AC). In terms of the total phenol obtained by the Folin-Ciocalteu colorimetric method, Mollar de Elche 2020 extracts reported the highest TPC content of 12.341 µmol GAE/g. Results revealed that the Mollar de Elche and Wonderful 2020 peel extracts demonstrated the highest TPC and AC. Furthermore, AC results indicated that the peel extracts displayed higher AC than the male flower extract due to the high punicalagin content detected by UPLC analysis. The antimicrobial activity testing revealed that the Wonderful and G1 2020 peel extracts resulted active against Escherichia coli, while all extracts exhibited promising anticandidal activity. Additionally, the cytocompatibility was evaluated in keratinocytes HaCaT cells by testing concentrations of pomegranate extracts ranging from 0.15 to 5.00 mg/mL. Extracts were non-toxic for the cells in the tested concentration range. The acquired results may help exploit pomegranate agri-food waste products provided by the Marche region's short supply chain for their use as an antimicrobial and antioxidant booster in the formulation of cosmetic products.

Acrylamide formation and antioxidant activity in coffee during roasting - A systematic study.

The aim of this study was to investigate the effect of the coffee roasting process on both toxic and some beneficial antioxidant compounds, applying a systematic and broad approach. Arabica and Robusta green coffee beans were roasted in a lab-scale roaster for different times in order to achieve five roasting degrees (from light to dark) and to assess the evolution of acrylamide (AA), trigonelline, nicotinic acid and caffeoylquinic acids contents (determined by HPLC) as well as antioxidant activity (evaluated by Folin-Ciocalteu, FRAP, DPPH, ABTS assays). The results confirmed that the AA levels and antioxidant activity reached a maximum in the first coffee roasting degrees and then decreased prolonging the heating process, both in Arabica and Robusta samples. Nevertheless, the thermal reduction observed was greater for AA compared to antioxidant activity, which was only slightly reduced due to the balance between the degradation and the neoformation of antioxidant compounds.

Quantification of 17 Endogenous and Exogenous Steroidal Hormones in Equine and Bovine Blood for Doping Control with UHPLC-MS/MS.

A simple and fast analytical method able to simultaneously identify and quantify 17 endogenous and exogenous steroidal hormones was developed in bovine and equine blood using UHPLC-MS/MS. A total amount of 500 µL of sample was deproteinized with 500 µL of a mixture of methanol and zinc sulfate and evaporated. The mixture was reconstituted with 50 µL of a solution of 25% methanol and injected in the UHPLC-MS/MS triple quadrupole. The correlation coefficients of the calibration curves of the analyzed compounds were in the range of 0.9932-0.9999, and the limits of detection and quantification were in the range of 0.023-1.833 and 0.069-5.5 ppb, respectively. The developed method showed a high sensitivity and qualitative aspects allowing the detection and quantification of all steroids in equine and bovine blood. Moreover, the detection limit of testosterone (50 ppt) is half of the threshold admitted in plasma (100 ppt). Once validated, the method was used to quantify 17 steroid hormones in both bovine and equine blood samples. The primary endogenous compounds detected were corticosterone (range 0.28-0.60 ppb) and cortisol (range 0.44-10.00 ppb), followed by androstenedione, testosterone and 11-deoxycortisol.