Saffron and retinal neurodegenerative diseases: Relevance of chemical composition.

Saffron is an ancient spice largely used in traditional medicine. It has been found to be effective in treatment of retinal neurodegenerative diseases like age-related macular degeneration and Stargardt. In the present manuscript, it is shown that saffron's neuroprotective power is strongly related to the bioactivity of all its chemical components. Nuclear magnetic resonance spectroscopy and "in vitro" experiments confirm the relevance of crocins for saffron efficacy. These results underline the importance of strictly defining the chemical composition of the natural compounds in saffron to optimize their effectiveness in the treatment of diseases.

Comprehensive Extraction and Chemical Characterization of Bioactive Compounds in Tepals of Crocus sativus L.

Crocus sativus L. is largely cultivated because it is the source of saffron, a well-appreciated and valued spice, not only for its culinary use but also because of its significant biological activities. Stigmas are the main product obtained from flowers, but in addition, tepals, largely considered a waste product, represent a big source of flavonoids and anthocyanins. This study aimed to delve into the phytochemical composition of saffron tepals and investigate whether the composition was influenced by the extraction technique while investigating the main analytical techniques most suitable for the characterization of tepal extracts. The research focuses on flavonoids, a class of secondary metabolites, and their health benefits, including antioxidant, anti-inflammatory, and anticancer properties. Flavonoids occur as aglycones and glycosides and are classified into various classes, such as flavones, flavonols, and flavanones. The most abundant flavonoids in tepals are kaempferol glycosides, followed by quercetin and isorhamnetin glycosides. Overall, this review provides valuable insights into the potential uses of tepals as a source of bioactive compounds and their applications in various fields, promoting a circular and sustainable economy in saffron cultivation and processing.

NMR-Based Metabolomic Study on Phaseolus vulgaris Flour Fermented by Lactic Acid Bacteria and Yeasts.

In recent years, fermented foods have attracted increasing attention due to their important role in the human diet, since they supply beneficial health effects, providing important sources of nutrients. In this respect, a comprehensive characterization of the metabolite content in fermented foods is required to achieve a complete vision of physiological, microbiological, and functional traits. In the present preliminary study, the NMR-based metabolomic approach combined with chemometrics has been applied, for the first time, to investigate the metabolite content of Phaseolus vulgaris flour fermented by different lactic acid bacteria (LAB) and yeasts. A differentiation of microorganisms (LAB and yeasts), LAB metabolism (homo- and heterofermentative hexose fermentation), LAB genus (Lactobacillus, Leuconostoc, and Pediococcus), and novel genera (Lacticaseibacillus, Lactiplantibacillus, and Lentilactobacillus) was achieved. Moreover, our findings showed an increase of free amino acids and bioactive molecules, such as GABA, and a degradation of anti-nutritional compounds, such as raffinose and stachyose, confirming the beneficial effects of fermentation processes and the potential use of fermented flours in the production of healthy baking foods. Finally, among all microorganisms considered, the Lactiplantibacillus plantarum species was found to be the most effective in fermenting bean flour, as a larger amount of free amino acids were assessed in their analysis, denoting more intensive proteolytic activity.

NMR Characterization of Lignans.

Lignans are particularly interesting secondary metabolites belonging to the phenyl-propanoid biosynthetic pathway. From the structural point of view, these molecules could belong to the aryltetralin, arylnaphtalene, or dibenzylbutyrolactone molecular skeleton. Lignans are present in different tissues of plants but are mainly accumulated in seeds. Extracts from plant tissues could be characterized by using the NMR-based approach, which provides a profile of aromatic molecules and detailed structural information for their elucidation. In order to improve the production of these secondary metabolites, elicitors could effectively stimulate lignan production. Several plant species are considered in this review with a particular focus on Linum species, well recognized as the main producer of lignans.

New Insight into Justicidin B Pathway and Production in Linum austriacum.

Lignans are the main secondary metabolites synthetized by Linum species as plant defense compounds but they are also valuable for human health, in particular, for novel therapeutics. In this work, Linum austriacum in vitro cultures, cells (Cc), adventitious roots (ARc) and hairy roots (HRc) were developed for the production of justicidin B through elicitation with methyl jasmonate (MeJA) and coronatine (COR). The performances of the cultures were evaluated for their stability, total phenols content and antioxidant ability. NMR was used to identify justicidin B and isojusticidin B and HPLC to quantify the production, highlighting ARc and HRc as the highest productive tissues. MeJA and COR treatments induced the synthesis of justicidin B more than three times and the synthesis of other compounds. RNA-sequencing and a de novo assembly of L. austriacum ARc transcriptome was generated to identify the genes activated by MeJA. Furthermore, for the first time, the intracellular localization of justicidin B in ARc was investigated through microscopic analysis. Then, HRc was chosen for small-scale production in a bioreactor. Altogether, our results improve knowledge on justicidin B pathway and cellular localization in L. austriacum for future scale-up processes.

Enhanced Aryltetralin Lignans Production in Linum Adventi-Tious Root Cultures.

Lignans are the main secondary metabolites synthetized by Linum species as plant defense molecules. They are also valuable for human health, in particular, for their potent antiviral and antineoplastic properties. In this study, the adventitious root cultures of three Linum species (L. flavum, L. mucronatum and L. dolomiticum) were developed to produce aryltetralin lignans. The effect of two elicitors, methyl jasmonate and coronatine, on aryltetralin lignans production was also evaluated. The adventitious root cultures from L. dolomiticum were obtained and analyzed for the first time and resulted as the best producer for all the aryltetralins highlighted in this system: Podophyllotoxin, 6-methoxypodophyllotoxin and 6-methoxypodophyllotoxin-7-O-ß-glucoside, the last showing a productivity of 92.6 mg/g DW. The two elicitors differently affected the production of the 6-methoxypodophyllotoxin and 6-methoxypodophyllotoxin-7-O-ß-glucoside.

NMR Lipid Profile of Milk from Alpine Goats with Supplemented Hempseed and Linseed Diets.

The supplementation of goat diets with natural products to obtain milk with nutraceutical components is a common practice. In these last years, the influence of supplementation of specifically designed diets has been studied with different analytical tools in order to explore possible beneficial effects in human consumption of animal milk and milk-derived products. In this study, the lipid fraction of milk from Alpine goats undergoing different dietary regimens was studied by 1H-NMR spectroscopy. Alpine goats were fed with linseed or hempseed supplements, and after 14 weeks of treatment, milk was collected and analyzed. Results showed that feeding diets supplemented with seeds positively affected the fatty acid composition with a pronounced increase in unsaturated fatty acids for both diets compared to a control diet. Specifically, linolenic acid content was more than doubled for linseed diet compared with the hempseed and control groups, while linoleic acid greatly increased only upon hempseed supplementation. However, a number of conjugated linoleic acid (CLA) isomers and higher levels of fatty acids with trans configuration were found in supplemented diets, particularly in the linseed diet.

The potentiality of NMR-based metabolomics in food science and food authentication assessment.

In the last years, there was an increasing interest on nuclear magnetic resonance (NMR) spectroscopy, whose applications experienced an exponential growth in several research fields, particularly in food science. NMR was initially developed as the elective technique for structure elucidation of single molecules and nowadays is playing a dominant role in complex mixtures investigations. In the era of the "omics" techniques, NMR was rapidly enrolled as one of the most powerful methods to approach metabolomics studies. Its use in analytical routines, characterized by rapid and reproducible measurements, would provide the identification of a wide range of chemical compounds simultaneously, disclosing sophisticated frauds or addressing the geographical origin, as well as revealing potential markers for other authentication purposes. The great economic value of high-quality or guaranteed foods demands highly detailed characterization to protect both consumers and producers from frauds. The present scenario suggests metabolomics as the privileged approach of modern analytical studies for the next decades. The large potentiality of high-resolution NMR techniques is here presented through specific applications and using different approaches focused on the authentication process of some foods, like tomato paste, saffron, honey, roasted coffee, and balsamic and traditional balsamic vinegar of Modena, with a particular focus on geographical origin characterization, ageing determination, and fraud detection.

Cell Suspensions of Cannabis sativa (var. Futura): Effect of Elicitation on Metabolite Content and Antioxidant Activity.

Cannabis sativa L. is one of the most-studied species for its phytochemistry due to the abundance of secondary metabolites, including cannabinoids, terpenes and phenolic compounds. In the last decade, fiber-type hemp varieties have received interest for the production of many specialized secondary metabolites derived from the phenylpropanoid pathway. The interest in these molecules is due to their antioxidant activity. Since secondary metabolite synthesis occurs at a very low level in plants, the aim of this study was to develop a strategy to increase the production of such compounds and to elucidate the biochemical pathways involved. Therefore, cell suspensions of industrial hemp (C. sativa L. var. Futura) were produced, and an advantageous elicitation strategy (methyl jasmonate, MeJA) in combination with precursor feeding (tyrosine, Tyr) was developed. The activity and expression of phenylalanine ammonia-lyase (PAL) and tyrosine aminotransferase (TAT) increased upon treatment. Through 1H-NMR analyses, some aromatic compounds were identified, including, for the first time, 4-hydroxyphenylpyruvate (4-HPP) in addition to tyrosol. The 4-day MeJA+Tyr elicited samples showed a 51% increase in the in vitro assay (2,2-diphenyl-1-picrylhydrazyl, DPPH) radical scavenging activity relative to the control and a 80% increase in the cellular antioxidant activity estimated on an ex vivo model of human erythrocytes. Our results outline the active metabolic pathways and the antioxidant properties of hemp cell extracts under the effect of specific elicitors.

Integrated analytical methodology to investigate bioactive compounds in Crocus sativus L. flowers.

INTRODUCTION: The increasing interest on Crocus sativus L. over the last decades is caused by its potential employment as a source of biologically active molecules, endowed with antioxidant and nutraceutical properties. These molecules are present mainly in stigmas and tepals, these last generally considered as byproducts. OBJECTIVE: To characterise bioactive compounds in stigmas, stamens, and tepals of Crocus sativus L. for quality, cross-contamination of tissues or fraudulent addition, joining spectroscopic and chromatographic techniques. METHODOLOGY: Fourier transform infrared (FT-IR) and Raman spectroscopies were initially employed, being very rapid in response; volatiles were more appropriately investigated by gas chromatography with mass spectrometry (GC-MS), while finally nuclear magnetic resonance (NMR) and high-performance liquid chromatography with a diode array detector (HPLC-DAD) were adopted for a more thorough characterisation of secondary metabolites. NMR was also used to investigate the anthocyanins content in tepals upon acid extraction. RESULTS: The results obtained highlighted the drying method as the dominant factor affecting the content of volatile constituents and contributing to the quality of saffron, while only slight differences were observed in the most abundant metabolites of stigmas, as well as in the anthocyanin content of tepals. In particular, for the first time, delphinidin and petunidin were detected by NMR in this latter tissue. CONCLUSION: The integrated analytical methodology here proposed, allowed to achieve a deeper level of compositional and structural details of secondary metabolites in Crocus sativus L. flowers.

Synthesis of Clickable Coating Polymers by Postpolymerization Modification: Applications in Microarray Technology.

In this paper, we report on the postpolymerization modification (PPM) of a polymer to introduce new functionalities that enable click chemistry reactions for microarray applications. The parent polymer, named copoly(DMA-NAS-MAPS), is composed of N,N-dimethylacrylamide (DMA), a monomer that self-adsorbs onto different materials through weak interactions such as hydrogen bonding or van der Waals forces, 3-(trimethoxysilyl)propyl methacrylate (MAPS) that strengthens the stability of the coating through the formation of covalent bonds with siloxane groups on the surface to be coated, and N-acryloyloxysuccinimide (NAS), an active ester group, highly reactive toward nucleophiles, which enables bioprobe immobilization. This copolymer has been widely exploited to coat surfaces for microarray applications but exhibits some limitations because of the potential hydrolysis of the active ester (NHS ester). The degradation of the NHS ester hampers the use of this coating in some situations, for example, when probe immobilization cannot be accomplished through a microspotting situation, but in large volumes, for example, in microchannel derivatization or micro-/nanoparticle functionalization. To overcome the limitations of NHS esters, we have developed a family of polymers that originate from the common copolymer precursor, by reacting the active ester contained in the polymer chain with a bifunctional amine. In particular, the functional groups introduced in the polymer using PPM enable click chemistry reactions such as azide/alkyne or thiol/maleimide "click" reactions, with suitably modified biomolecules. The advantages of such reactions are quantitative yields, orthogonality of functional groups, and insensitivity of the reaction to pH. The new click functionalities, inserted with quantitative yields, improve the stability of the coating, enabling the attachment of biomolecules directly from a solution and avoiding the spotting of reduced volumes (pL) of probes. Finally, we have demonstrated the applicability of the click surfaces in a highly effective solid-phase PCR for the genotyping of the G12D KRAS mutation.

On the Traceability of Commercial Saffron Samples Using ¹H-NMR and FT-IR Metabolomics.

In previous works on authentic samples of saffron of known history (harvest and processing year, storage conditions, and length of time) some biomarkers were proposed using both FT-IR and NMR metabolomics regarding the shelf life of the product. This work addresses the difficulties to trace back the "age" of commercial saffron samples of unknown history, sets a limit value above which these products can be considered substandard, and offers a useful tool to combat saffron mislabeling and fraud with low-quality saffron material. Investigations of authentic and commercial saffron samples of different origin and harvest year, which had been stored under controlled conditions for different lengths of time, allowed a clear-cut clustering of samples in two groups according to the storage period irrespectively of the provenience. In this respect, the four-year cut off point proposed in our previous work assisted to trace back the "age" of unknown samples and to check for possible mislabeling practices.

Performance Assessment in Fingerprinting and Multi Component Quantitative NMR Analyses.

An interlaboratory comparison (ILC) was organized with the aim to set up quality control indicators suitable for multicomponent quantitative analysis by nuclear magnetic resonance (NMR) spectroscopy. A total of 36 NMR data sets (corresponding to 1260 NMR spectra) were produced by 30 participants using 34 NMR spectrometers. The calibration line method was chosen for the quantification of a five-component model mixture. Results show that quantitative NMR is a robust quantification tool and that 26 out of 36 data sets resulted in statistically equivalent calibration lines for all considered NMR signals. The performance of each laboratory was assessed by means of a new performance index (named Qp-score) which is related to the difference between the experimental and the consensus values of the slope of the calibration lines. Laboratories endowed with a Qp-score falling within the suitable acceptability range are qualified to produce NMR spectra that can be considered statistically equivalent in terms of relative intensities of the signals. In addition, the specific response of nuclei to the experimental excitation/relaxation conditions was addressed by means of the parameter named NR. NR is related to the difference between the theoretical and the consensus slopes of the calibration lines and is specific for each signal produced by a well-defined set of acquisition parameters.

Performance of a novel sieving matrix of poly(vinyl alcohol)/acrylamide copolymer in electrophoretic separations of high molecular weight proteins from red cell membrane.

The analysis of high molecular weight (HMW) proteins from complex mixtures is still a challenge in proteomics. This work introduces a novel hydrogel obtained by the copolymerization of an allyl-PVA derivative with acrylamide and bisacrylamide and applies this matrix to the electrophoretic separation of HMW proteins. By inducing gelation of polyacrylamide in the presence of variable amounts of allyl-PVA, it is possible to control and vary the average gel porosity. This gel is easy to produce and handle and offers the advantage of being highly mechanically resistant and macroporous. The new matrix was tested in mono-dimensional separations of complex protein mixtures extracted from red cell membranes with different detergents. The improved performance of this macroporous matrix allowed to identify new proteins by MS and immunoblot analysis using specific antibodies. In particular, the resolution of proteins ranging in size between 97 and 279 kDa was greatly improved here compared to standard polyacrylamide gels, suggesting that this matrix can be a useful tool in routine analysis of HMW proteins in cell biology.

Early phenylpropanoid biosynthetic steps in Cannabis sativa: link between genes and metabolites.

Phenylalanine ammonia-lyase (PAL), Cinnamic acid 4-hydroxylase (C4H) and 4-Coumarate: CoA ligase (4CL) catalyze the first three steps of the general phenylpropanoid pathway whereas chalcone synthase (CHS) catalyzes the first specific step towards flavonoids production. This class of specialized metabolites has a wide range of biological functions in plant development and defence and a broad spectrum of therapeutic activities for human health. In this study, we report the isolation of hemp PAL and 4CL cDNA and genomic clones. Through in silico analysis of their deduced amino acid sequences, more than an 80% identity with homologues genes of other plants was shown and phylogenetic relationships were highlighted. Quantitative expression analysis of the four above mentioned genes, PAL and 4CL enzymatic activities, lignin content and NMR metabolite fingerprinting in different Cannabis sativa tissues were evaluated. Furthermore, the use of different substrates to assay PAL and 4CL enzymatic activities indicated that different isoforms were active in different tissues. The diversity in secondary metabolites content observed in leaves (mainly flavonoids) and roots (mainly lignin) was discussed in relation to gene expression and enzymatic activities data.

Linum lewisii Adventitious and Hairy-Roots Cultures as Lignan Plant Factories.

Plants synthesize specific secondary metabolites for survival, reproduction, environmental resilience, and defense. Among them, lignans are a class of polyphenols with several bioactive properties: chemopreventive, anti-inflammatory, antiviral, and antioxidant. These compounds are often extracted from field-grown plants with very low yields. To overcome these constraints, in vitro tissue cultures provide a tool to optimize large-scale production. Moreover, the use of elicitation to increase secondary metabolite production is gaining importance. The aim of this work was to develop adventitious (ARL) and hairy roots (HRL) from Linum lewisi, a species able to synthesize arylnaphthalene lignans such as justicidin B. The ARL and HRL were obtained for the first time and characterized for their phenol content, antioxidant activity, and the production of justicidin B after treatments with several elicitors and precursor feeding. Through NMR spectroscopy, other four lignans were highlighted and identified in the roots extracts. A pilot-scale bioreactor was adopted to assess the suitability of the developed root cultures for future large-scale production. The ARL and HRL cultures showed a justicidin B production higher than other Linum species cultures described up to now (75.8 mg/L and 82.2 g/L), and the production more than doubled after elicitation with MeJA.

Sequential Infiltration Synthesis of Al2O3 in Biodegradable Polybutylene Succinate: Characterization of the Infiltration Mechanism.

The introduction of inorganic materials into biopolymers has been envisioned as a viable option to modify the optical and structural properties of these polymers and promote their exploitation in different application fields. In this work, the growth of Al2O3 in freestanding ∼30-µm-thick poly(butylene succinate) (PBS) films by sequential infiltration (SIS) at 70 °C via trimethylaluminum (TMA) and H2O precursors was investigated for the first time. The incorporation of Al2O3 into the PBS matrix was clearly demonstrated by XPS analysis and SEM-EDX cross-sectional images showing a homogeneous Al2O3 distribution inside the PBS films. Raman measurements on infiltrated freestanding PBS show a reduction of the signal related to the ester carbonyl group as compared to pristine freestanding PBS films. Accordingly, FTIR and NMR characterization highlighted that the ester group is involved in polymer-precursor interaction, leading to the formation of an aliphatic group and the concomitant rupture of the main polymeric chain. Al2O3 mass uptake as a function of the number of SIS cycles was studied by infiltration in thin PBS films spin-coated on Si substrates ranging from 30 to 70 nm. Mass uptake in the PBS films was found to be much higher than in standard poly(methyl methacrylate) (PMMA) films, under the same process conditions. Considering that the density of reactive sites in the two polymers is roughly the same, the observed difference in Al2O3 mass uptake is explained based on the different free volume of these polymers and the specific reaction mechanism proposed for PBS. These results assessed the possibility to use SIS as a tool for the growth of metal oxides into biopolymers, paving the way to the synthesis of organic-inorganic hybrid materials with tailored characteristics.

Hydration studies on the archaeal protein Sso7d using NMR measurements and MD simulations.

BACKGROUND: How proteins approach surrounding molecules is fundamental to our understanding of the specific interactions that occur at the surface of proteins. The enhanced surface accessibility of small molecules such as organic solvents and paramagnetic probes to protein binding sites has been observed; however, the molecular basis of this finding has not been fully established. Recently, it has been suggested that hydration dynamics play a predominant role in controlling the distribution of hot spots on surface of proteins. RESULTS: In the present study, the hydration of the archaeal multifunctional protein Sso7d from Solfolobus solfataricus was investigated using a combination of computational and experimental data derived from molecular dynamics simulations and ePHOGSY NMR spectroscopy. CONCLUSIONS: We obtained a convergent protein hydration landscape that indicated how the shape and stability of the Sso7d hydration shell could modulate the function of the protein. The DNA binding domain overlaps with the protein region involved in chaperon activity and this domain is hydrated only in a very small central region. This localized hydration seems to favor intermolecular approaches from a large variety of ligands. Conversely, high water density was found in surface regions of the protein where the ATP binding site is located, suggesting that surface water molecules play a role in protecting the protein from unspecific interactions.

A bi-functional polymeric coating for the co-immobilization of proteins and peptides on microarray substrates.

The analytical performance of the microarray technique in screening the affinity and reactivity of molecules towards a specific target, is highly affected by the coupling chemistry adopted to bind probes to the surface. However, the surface functionality limits the biomolecules that can be attached to the surface to a single type of molecule, thus forcing the execution of separate analyses to compare the performance of different species in recognizing their targets. Here we introduce a new N, N-dimethylacrylamide-based polymeric coating, bearing simultaneously different functionalities (N-acryloyloxysuccinimide and azide groups) to allow an easy and straightforward method to co-immobilize proteins and oriented peptides on the same substrate. The bi-functional copolymer has been obtained by partial post polymerization modification of the functional groups of a common precursor. A NMR characterization of the copolymer was conducted to quantify the percentage of NAS that has been transformed into azido groups. The polymer was used to coat surfaces onto which both native antibodies and alkyne modified peptides were immobilized, to perform the phenotype characterization of extracellular vesicles (EVs). This strategy represents a convenient method to reduce the number of analysis, thus possible systematic or random errors, besides offering a drastic shortage in time, reagents and costs.

Polyurethane-Based Composites: Effects of Antibacterial Fillers on the Physical-Mechanical Behavior of Thermoplastic Polyurethanes.

The challenge to manufacture medical devices with specific antibacterial functions, and the growing demand for systems able to limit bacterial resistance growth, necessitates the development of new technologies which can be easily produced at an industrial level. The object of this work was the study and the development of silver, titanium dioxide, and chitosan composites for the realization and/or implementation of biomedical devices. Thermoplastic elastomeric polyurethane was selected and used as matrix for the various antibacterial functions introduced during the processing phase (melt compounding). This strategy was employed to directly incorporate antimicrobial agents into the main constituent material of the devices themselves. With the exception of the composite filled with titanium dioxide, all of the other tested composites were shown to possess satisfactory mechanical properties. The best antibacterial effects were obtained with all the composites against Staphylococcus aureus: viability was efficiently inhibited by the prepared materials in four different bacterial culture concentrations.