Characterization of Phenolic Profile in Milk Obtained by Ewes Fed Grape Pomace: Reflection on Antioxidant and Anti-Inflammatory Status.

The aim of the present work was to evaluate if the use of grape pomace (GP) in the feeding of dairy ewes can improve the content of phenolic compounds (PCs) in the milk and affect the anti-inflammatory and antioxidative status of the milk. For this purpose, 46 ewes were randomly assigned to two groups of 23 animals each: a control group (Ctrl) that received a standard diet and an experimental group (GP+), whose diet was been formulated with 10% GP on a dry matter (DM) basis. At the end of the 60 days of the trial, from 10 ewes selected randomly from each group, individual milk samples were collected and analyzed for the identification and the quantification of phenolic compounds through an ultra-high-performance liquid chromatography system, and milk anti-inflammatory and antioxidative status were evaluated by enzyme-linked immunosorbent assay, determining the activity of GPx and CAT and the levels of IL-1 and TNFα. In addition, gelatinolytic activity of Type IV collagenases (MMP-2/MMP-9) was evaluated by the fluorometric method and zymographic approach. The results obtained showed that the diet with GP affects the phenolic profile of milk, inducing milk enrichment of phenolic compounds without, however, having a significant impact on milk antioxidant and inflammatory status. However, a lower activity of MMP-9 was found in GP+ milk. The use of the molecular docking approach showed the ability of luteolin to approach the catalytic pocket of the enzyme, interfering with the recruitment of the substrate, and therefore, slowing down their hydrolytic activity.

Chemical characterization and evaluation of antioxidant activity from different cultivars of Cannabis sativa L. of Abruzzo's region.

In this work, the chemical composition and the antioxidant evaluation of the inflorescences from 12 Cannabis sativa L. monoecious cultivars (Carmagnola Lemon CL, Ferimon F, Gran Sasso Kush GSK, Antal A, Carmagnola C, Kompolti K, Futura 75 F75, Villanova V, Tiborzallasi T, Finola FL, Kc Virtus KV and Pineapple P) cultivated at the same condition, were investigated. GC-MS analysis was carried out to evaluate the volatile fraction, while HPLC-MS/MS was used for cannabinoids and polyphenolic compounds. The evaluation of antioxidant activity was carried out using ABTS*+, Trolox equivalence antioxidant capacity (TEAC), ferric reducing antioxidant property (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH*) assays in vitro. The obtained data, demonstrated that each cultivar has a characteristic chemical profile, with highest antioxidant capacity for CL, F75, GSK and F. Based on the in vitro antioxidant activity the plant extracts can be considered as promising candidates for different applications in food field.

Starch-based sustainable hydrogel loaded with Crocus sativus petals extract: A new product for wound care.

The aim of the present study was to valorize Crocus sativus petals, the main waste deriving from saffron stigma harvesting, as source of bioactive molecules to be used in health field. Three different dry extracts were prepared by eco-friendly methods (maceration and ultrasound bath assisted maceration) using saffron petals as raw material and ethanol 70 % either ethanol 96 % as extraction solvents. A preliminary evaluation of the antioxidant activity (measured by ABTS*+, DPPH* and FRAP) highlighted that the most suitable extraction solvent is represented by ethanol 70 %. By in vitro studies on keratinocytes emerged that the extract obtained by maceration (rich in gallic and chlorogenic acids) stimulates their growth in a safe concentration range (0.02-0.4 mg/mL) suggesting a potential application in skin diseases such as superficial wounds. Due to the low manageability, the extract was firstly supported on corn starch powder particles and then formulated as starch gel. The obtained formulation showed both suitable rheological properties and spreadability necessary for an easy and pain free application on damaged skin. Moreover, in vitro microbiological studies of starch gel demonstrated antimicrobial activity toward S. epidermidis and self-preserving capacity.

Effect of Wheat Crop Nitrogen Fertilization Schedule on the Phenolic Content and Antioxidant Activity of Sprouts and Wheatgrass Obtained from Offspring Grains.

This work was aimed at investigating the effects of rate and timing of nitrogen fertilization applied to a maternal wheat crop on phytochemical content and antioxidant activity of edible sprouts and wheatgrass obtained from offspring grains. We hypothesized that imbalance in N nutrition experienced by the mother plants translates into transgenerational responses on seedlings obtained from the offspring seeds. To this purpose, we sprouted grains of two bread wheat cultivars (Bologna and Bora) grown in the field under four N fertilization schedules: constantly well N fed with a total of 300 kg N ha-1; N fed only very early, i.e., one month after sowing, with 60 kg N ha-1; N fed only late, i.e., at initial shoot elongation, with 120 kg N ha-1; and unfertilized control. We measured percent germination, seedling growth, vegetation indices (by reflectance spectroscopy), the phytochemical content (total phenols, phenolic acids, carotenoids, chlorophylls), and the antioxidant activity (by gold nanoparticles photometric assay) of extracts in sprout and wheatgrass obtained from the harvested seeds. Our main finding is that grains obtained from crops subjected to late N deficiency produced wheatgrass with much higher phenolic content (as compared to the other N treatments), and this was observed in both cultivars. Thus, we conclude that late N deficiency is a stressing condition which elicits the production of phenols. This may help counterbalance the loss of income related to lower grain yield in crops subjected to such an imbalance in N nutrition.

Predictive Multi Experiment Approach for the Determination of Conjugated Phenolic Compounds in Vegetal Matrices by Means of LC-MS/MS.

Polyphenols (PCs) are a numerous class of bioactive molecules and are known for their antioxidant activity. In this work, the potential of the quadrupole/linear ion trap hybrid mass spectrometer (LIT-QqQ) was exploited to develop a semi-untargeted method for the identification of polyphenols in different food matrices: green coffee, Crocus sativus L. (saffron) and Humulus lupulus L. (hop). Several conjugate forms of flavonoids and hydroxycinnamic acid were detected using neutral loss (NL) as a survey scan coupled with dependent scans with enhanced product ion (EPI) based on information-dependent acquisition (IDA) criteria. The presented approach is focused on a specific class of molecules and provides comprehensive information on the different conjugation models that are related to specific base molecules, thus allowing a quick and effective identification of all possible combinations, such as mono-, di-, or tri-glycosylation or another type of conjugation such as quinic acid esters.

Cannabinoid Profile in Cannabis sativa L. Samples by Means of LC-MRM/IDA/EPI Analysis: A New Approach for Cultivar Classification.

A new multitarget screening procedure for 36 cannabinoids in 12 Cannabis sativa L. cultivars (hemp) was developed using multiple reaction monitoring (MRM) coupled with an enhanced product ion (EPI) scan in an information-dependent acquisition (IDA) experiment, which can be performed by means of high-performance liquid chromatography-mass spectrometry (HPLC-MS)/MS analysis. The MRM-IDA-EPI experiment was used for the analysis of hemp samples and the identification of the compounds of interest. It was performed through the comparison of EPI spectra with literature data and with the in-house library. The results, processed by multivariate statistical analysis, showed an accurate classification of the 12 C. sativa cultivars, emphasizing the synergic contribution of the new cannabinoids recently discovered and showing how the traditional classification based on a common cannabinoid is limiting.

Hazelnut Shells as Source of Active Ingredients: Extracts Preparation and Characterization.

Hazelnut shells represent a waste material (about 42% of the total biomass) deriving from hazelnut harvest. These are mainly used as a heating source; however, they represent an interesting source of polyphenols useful in health field. The impact on phenolic profile and concentrations of hazelnut shell extracts obtained by three extraction methods (maceration, ultrasonic bath, and high-power ultrasonic), as well as temperature, extraction time, and preventive maceration, was studied. The prepared extracts were characterized in terms of chemical composition, antioxidant and antimicrobial activities. Eighteen different phenolic compounds were identified and quantified by chemical analysis and gallic acid was the most abundant in all the extracts analyzed. Other relevant compounds were chlorogenic acid, protocatechuic acid and catechin. Preventive maceration had a positive effect on the extraction of different types of compounds regardless of the method performed. Application of the high-power ultrasonic method had different effects, either positive or negative, depending on the type of compound and extraction time. All the prepared extracts showed antioxidant activity especially those prepared by maceration, and many of them were able to inhibit the growth of both B. subtilis and B. cereus.

Targeted and semi-untargeted determination of phenolic compounds in plant matrices by high performance liquid chromatography-tandem mass spectrometry.

In this work two different acquisition approaches were used for the quantification and/or tentative identification of phenolic compounds (PCs) in plant matrices by HPLC-MS/MS. A targeted approach, based on MRM acquisition mode, was used for the identification and quantification of a list of target analytes by comparison with standards; a semi-targeted approach was also developed by the precursor ion scan and neutral loss for the tentative identification of compounds not included in the target list. Analysis of phenolic content in three different plant matrices (curry leaves, hemp and blueberry) was carried out. The extraction and clean-up steps were set up according to the characteristics of the sample allowing to minimize the interfering compounds present in such complex matrices, as proved by the low matrix effect obtained (<16%) and recovery values ranging from 45% to 98% for all the analytes. This approach provided a sensitive and robust quantitative analysis of the target compounds with LOQs between 0.0002 and 0.05 ng mg-1, which allowed the identification and quantification of several hydroxycinnamic and hydroxybenzoic acids, in addition to numerous flavonoids in all three matrices. Furthermore, different moieties were considered as neutral losses or as precursor ions in semi-targeted MS/MS approach, providing the putative identification of different glycosylated forms of flavonoids, such as luteolin-galactoside and diosmin in all three matrices, while apigenin-glucuronide was detected in hemp and quercetin-glucuronide in blueberry. A further study was carried out by MS3, allowing the discrimination of compounds with similar aglycones, such as luteolin and kaempferol.

µSPE followed by HPLC-MS/MS for the determination of series D and E resolvins in biological matrices.

The critical role of acute inflammatory processes is recognized in many chronic diseases; a key point in molecular mechanisms of acute inflammation resolution is represented by a new group of pro-resolving lipid mediators that include distinct families of molecules: lipoxins, resolvins, protectins and maresins, collectively termed "specialized pro-resolving mediators" (SPMs). In particular, resolvins are active in the picogram to nanogram dose range, whereby they can directly modulate a plethora of anti-inflammatory responses. The presented method proposes an analytical protocol able to extract and to quantify 6 different resolvins from 3 different matrices (plasma, cells and exudates). The method, validated according to the EMA guideline for bioanalysis, exhibited good precision (1%-20%) and accuracy (2%-20%). In particular, the combination of two different sample preparation techniques, Liquid-Liquid Extraction (LLE) and micro-Solid Phase Extraction (µSPE), applied for the first on this class of molecules, used for the extraction and clean-up respectively, led to high enrichment factor (20 fold) and consequently a high sensitivity (LOQ between 1 and 38 pg mL-1); moreover the validation data proved the versatility of µSPE as clean-up tool as it was capable to manage huge enrichment factor without negatively affect accuracy and precision of analysis.

Accelerated Extraction and Analysis of Ethyl Glucuronide in Hair by Means of Pressurized Liquid Extraction Followed by Liquid Chromatography-Tandem Mass Spectrometry Determination.

The measurement of ethyl glucuronide (EtG) in hair is an established practice to evaluate alcohol consumption habits of the donors; nevertheless, analytical variability has shown to be an important factor to be considered: measured EtG values can vary significantly as a consequence of analyte washout during decontamination, pulverization of samples, extraction solvent and incubation temperature. In the present study, we described a new method for automated hair decontamination and EtG extraction from the inner core of the hair by using pressurized liquid extraction (PLE), followed by solid-phase extraction (SPE) cleanup; validation was performed according to SWGTOX guidelines. The extraction efficiency of the new method was evaluated by comparing the results with those obtained by a validated and ISO/IEC 17025:2005 accredited method; an average positive difference of + 32% was observed when the extraction was performed by PLE. The effect of hair pulverization was also studied, and a good correlation between cut and milled hair was observed, implying that PLE allowed a highly efficient extraction of EtG from the inner keratin core of the hair, no matter if it has been cut or pulverized. Finally, to verify the results, paired aliquots of 27 real hair samples were analyzed with both PLE and a protocol optimized by design-of-experiment strategies planned to maximize the extraction yield; in this case, a comparable efficiency was observed, suggesting that exhaustive EtG extraction was obtained with both approaches. This finding opens new perspectives in the eligible protocols devoted to hair EtG analysis, in terms of speed, automation and reproducibility.