In Vitro Bioactivity of Astaxanthin and Peptides from Hydrolisates of Shrimp (Parapenaeus longirostris) By-Products: From the Extraction Process to Biological Effect Evaluation,as Pilot Actions for the Strategy “From Waste to Profit”

Non-edible parts of crustaceans could be a rich source of valuable bioactive compounds such as the carotenoid astaxanthin and peptides, which have well-recognized beneficial effects. These compounds are widely used in nutraceuticals and pharmaceuticals, and their market is rapidly growing, suggesting the need to find alternative sources. The aim of this work was to set up a pilot-scale protocol for the reutilization of by-products of processed shrimp, in order to address the utilization of this valuable biomass for nutraceutical and pharmaceuticals application, through the extraction of astaxanthin-enriched oil and antioxidant-rich protein hydrolysates. Astaxanthin (AST) was obtained using “green extraction methods,” such as using fish oil and different fatty acid ethyl esters as solvents and through supercritical fluid extraction (SFE), whereas bioactive peptides were obtained by protease hydrolysis. Both astaxanthin and bioactive peptides exhibited bioactive properties in vitro in cellular model systems, such as antioxidant and angiotensin I converting enzyme (ACE) inhibitory activities (IA). The results show higher astaxanthin yields in ethyl esters fatty acids (TFA) extraction and significant enrichment by short-path distillation (SPD) up to 114.80 ± 1.23 µg/mL. Peptide fractions of <3 kDa and 3–5 kDa exhibited greater antioxidant activity while the fraction 5–10 kDa exhibited a better ACE-IA. Lower-molecular-weight bioactive peptides and astaxanthin extracted using supercritical fluids showed protective effects against oxidative damage in 142BR and in 3T3 cell lines. These results suggest that “green” extraction methods allow us to obtain high-quality bioactive compounds from large volumes of shrimp waste for nutraceutical and pharmaceutical applications.     

Central fatigue and nycthemeral change of serum tryptophan and serotonin in the athletic horse

Background  

The serotonergic system is associated with numerous brain functions, including the resetting of the mammalian circadian clock. The synthesis and metabolism of 5-HT in the brain increases in response to exercise and is correlated with high levels of blood-borne tryptophan (TRP). The present investigation was aimed at testing the existence of a daily rhythm of TRP and 5-HT in the blood of athletic horses.     

Antioxidant and Antiproliferative Activity of <Emphasis Type="Italic">Diospyros lotus</Emphasis> L. Extract and Isolated Compounds

The object of the study was to determine the chemical composition of Diospyros lotus L. extract and their antioxidant and antiproliferative properties. Eight compounds were isolated from D. lotus and identified as gallic acid, methylgallate, ellagic acid, kaempferol, quercetin, myricetin, myricetin 3-O-β-glucuronide, and myricetin-3-O-α-rhamnoside. D. lotus extract tested in different in vitro systems (DPPH, ABTS, FRAP, and Fe²⁺ chelating activity assay) showed significant antioxidant activity. The potential antiproliferative properties of D. lotus extract and isolated compounds against nine human cancer cell lines such as COR-L23, CaCo-2, C32, ACHN, A375, A549, Huh-7D12, MCF-7, and LNCaP were investigated in vitro by SRB assay. D. lotus extract demonstrated the highest inhibitory activity against COR-L23 with an IC₅₀ value of 12.2 μg/ml. Among identified hydrolysable tannins, ellagic acid evidenced strong antiproliferative activity against both C32 and A375 cells with IC₅₀ values of 0.8 and 4.1 μg/ml, respectively. Interesting results were observed, also, with gallic acid that showed the highest cytotoxic activity against CaCo-2 (IC₅₀ 2.6 μg/ml). Overall, the results of this study suggest that D. lotus displays a good antioxidant activity and has antiproliferative effects. Both activities are related to identified phenolic compounds.     

Metabolic profiling and analysis of volatile composition of durum wheat semolina and pasta

Although pasta is generally not considered for its aromatic properties, some evidence proves that cereal flours release volatile compounds and they might have an effect on the aroma of the transformed products. This work reports on the characterization of the volatile components of semolina and pasta obtained from four durum wheat cultivars (Triticum durum Desf., cvs. PR22D89, Creso, Cappelli, Trinakria). Semolina samples were characterized through polar metabolite profiling and fatty acid analysis to identify potential precursors of the volatile components. The results show significant differences among the samples tested with cv. Trinakria characterized by the highest content of sugars and fatty acids. Volatile composition was investigated both in semolina and in cooked pasta using headspace solid-phase micro-extraction (HS-SPME) and identified by GC–MS. Thirty-five volatile compounds including aldehydes, ketones, alcohols, terpenes, esters, hydrocarbons and a furan were identified. Significant differences were observed between semolina and pasta samples in terms of composition and amount of the volatile compounds. During cooking an increase in aldehyde content, the appearance of ketones and a decrease in alcohol content were observed. Correlations between metabolites and volatiles demonstrate that the flavour of cooked pasta may differ significantly depending on the durum wheat cultivar employed.     

Characterization of Phaseolus vulgaris L. Landraces Cultivated in Central Italy

Eight Phaseolus vulgaris L. landraces cultivated on farm in marginal areas of Central Italy (Lazio region) were investigated in order to evaluate chemical composition of storage proteins and secondary metabolites fractions. The total protein content showed some differences among landraces; the maximum value was next to 30 g for 100 g of dry weight. The seed storage proteins were screened by polyacrylamide gel electrophoresis (SDS/PAGE): seven landraces exhibited phaseolin patterns type S, one landrace showed a phaseolin pattern type T. A morphological analysis of cotyledon parenchyma performed by scanning electron microscopy (SEM) revealed differences in size of starch granules. Moreover the polyphenolic composition was investigated using HPLC-APCI; from the methanol extracts a flavonoid, kaempferol, and a coumarin, 5,7-dimethoxycoumarin, were identified. To our knowledge, this is the first time that 5,7-dimethoxycoumarin has been reported in P. vulgaris seeds.     

In situ detection of tree root distribution and biomass by multi-electrode resistivity imaging

Traditional methods for studying tree roots are destructive and labor intensive, but available nondestructive techniques are applicable only to small scale studies or are strongly limited by soil conditions and root size. Soil electrical resistivity measured by geoelectrical methods has the potential to detect belowground plant structures, but quantitative relationships of these measurements with root traits have not been assessed. We tested the ability of two-dimensional (2-D) DC resistivity tomography to detect the spatial variability of roots and to quantify their biomass in a tree stand. A high-resolution resistivity tomogram was generated along a 11.75 m transect under an Alnus glutinosa (L.) Gaertn. stand based on an alpha-Wenner configuration with 48 electrodes spaced 0.25 m apart. Data were processed by a 2-D finite-element inversion algorithm, and corrected for soil temperature. Data acquisition, inversion and imaging were completed in the field within 60 min. Root dry mass per unit soil volume (root mass density, RMD) was measured destructively on soil samples collected to a depth of 1.05 m. Soil sand, silt, clay and organic matter contents, electrical conductivity, water content and pH were measured on a subset of samples. The spatial pattern of soil resistivity closely matched the spatial distribution of RMD. Multiple linear regression showed that only RMD and soil water content were related to soil resistivity along the transect. Regression analysis of RMD against soil resistivity revealed a highly significant logistic relationship (n = 97), which was confirmed on a separate dataset (n = 67), showing that soil resistivity was quantitatively related to belowground tree root biomass. This relationship provides a basis for developing quick nondestructive methods for detecting root distribution and quantifying root biomass, as well as for optimizing sampling strategies for studying root-driven phenomena.     

Regulation of mycorrhiza development in durum wheat by P fertilization: Effect on plant nitrogen metabolism

The aim of this work was to study the effect of arbuscular mycorrhizal fungus Glomus mosseae on growth and nitrogen (N) metabolism of durum wheat (Tritcum durum) under various P soil contents. The analyses were extended to macro and micronutrient tissue concentrations, nitrate reductase and glutamine synthetase activities, as well as protein, aminoacids, pyridine dinucleotides and adenine nucleotides. Arbuscular mycorrhiza increased wheat growth in soil in which P availability was low and nitrate was the dominant N form. The root colonization occurred at the highest level in plants grown in limiting soil P and was inversely related to soil P content. The micorrhizal wheat plants contained also the highest concentrations of macro (P, K, Ca, N) and micronutrients (Fe, Zn, Mn) as well as free amino acids, protein, NAD, NADP, AMP, ADP, ATP in roots and leaves. In particular, the micronutrient tissue concentrations (Zn, Mn) supported that mycorrhiza actively modulated their uptake limiting interferences and optimizing growth better than the plant roots, like a very efficient “rootstock”. Control plants grown at the highest soil P did not reach the same concentration as the mycorrhizal plants. Nitrate reductase activities in the roots of mycorrhizal plants were higher than in the control ones, while glutamine synthetase activities were highest in the leaves. Protein and amino acids concentrations, as well as AMP, ADP, ATP, NAD(P), and NAD(P)H were also higher than in the control. Among the free amino acids in the roots, the high levels of glutamine, asparagine, arginine, support the view that ammonium was transferred through the arbuscules to the root cells where it was re‐assimilated in the cortical cells, forming high N : C ratio‐amino acids. They were transferred to the leaves where all the other N compounds could be largely synthesized using the carbon skeletons supplied by photosynthesis.     

Comparison of blood and milk non-specific immune parameters in heifers after calving in relation to udder health

A practical protocol to study udder immune status in field conditions was planned with the aim to assess different non-specific immune parameters in milk samples from dairy heifers during the periparturient period. Five herds located in northern Italy were selected and overall 39 heifers were enrolled in the trial. Milk samples were taken at 7, 14, 21, 28, 45, 60, and 75 days after calving. The parameters assessed were N-acetyl-beta-glucosaminidase (NAGase), lysozyme, respiratory burst (RB), somatic cell counts (SCC) and serum protein profile. SCC and NAGase were higher in the first sampling after calving, while lysozyme showed large variations during the observation period without a definite trend. The levels of RB observed in the first two weeks after calving, even if lower, were not statistically different from the values observed in samples taken over the following weeks. This study confirmed that the levels of immune components in milk are different from what is observed at blood level in the same cow. A significant decrease in RB in milk polymorphonuclear leukocytes (PMN) post-calving was not observed; milk PMN from healthy cows showed low RB levels, while the values from infected quarters were significantly higher. Significant differences between healthy and infected animals were also observed for milk NAG, lactoglobulin and albumin. These data suggest that udder immune response could be influenced both by the cow immune status and by external factors such as pathogens and management. Therefore, the reduction in immune defences, particularly in heifers, is not unavoidable and methods to boost PMN activity should be explored.     

Expression of the KIT protein (CD117) in primary cutaneous mast cell tumors of the dog.

Thirty-one canine cutaneous masses, diagnosed as mast cell tumors (MCT) by histopathologic analysis, were used to evaluate the immunohistochemical pattern of expression of KIT protein (CD117), a type III tyrosine kinase protein involved in mast cell growth and differentiation. Lesions were graded as I (well differentiated), II (intermediate differentiation), or III (poorly differentiated) according to the following morphologic features: invasiveness, cellularity and cellular morphology, mitotic index, and stromal reaction. Immunohistochemical KIT expression was compared with histologic grade and some histomorphologic features (cell differentiation and nuclear grade) evaluated separately. A possible predictive role of biologic behavior in MCTs for KIT expression was also investigated. Immunohistochemical analysis revealed three different patterns of KIT expression: a cytoplasmic diffuse pattern, a membranous pattern with immunostaining located on the cell surface, and a cytoplasmic perinuclear pattern, where KIT expression was detected in the cytoplasm of the neoplastic mast cells, close to the nucleus. Statistical analysis showed a close relationship between different KIT immunohistochemical patterns and histologic grade (P < 0.00000), cell differentiation (P < 0.00000), and nuclear grade (P < 0.0024). According to Kaplan-Meier-estimated survival curves compared by survival analysis, KIT expression was significantly associated with survival time (P = 0.037) but not cancer-free interval (P = 0.50). Similar to other well-known histomorphological features, KIT expression is a useful parameter of malignancy in cutaneous MCTs. KIT expression also predicted the biological behavior of the tumors in this study.