Diversity of Pleurotus spp. (Agaricomycetes) and Their Metabolites of Nutraceutical and Therapeutic Importance.

Pleurotus spp. have been gaining popularity as a source for the creation of functional foods, nutraceuticals and novel pharmaceuticals. Despite Pleurotus is a specious genus including 208 legitimate species, only a few of them such as P. ostreatus are commercially accessible. The genetic and metabolic diversity of Pleurotus both at specific and subspecific level is therefore of main concern for many researchers. In addition to the conventional morphological approach, molecular and biochemical markers have been greatly contributing to investigate these issues. In this study, samples from six Pleurotus species (P. eryngii is represented by three varieties) were molecularly identified and the phylogeny was inferred to assess the relationships between the various taxa. Strains in pure culture obtained from 6 out of 7 species were cultivated as mycelium in vitro to investigate the metabolites by untargeted LC-MS/MS-based metabolomics. The results pointed out species-specific metabolite patterns and highlighted a clear difference between the P. eryngii group and P. ostreatus, although the latter appears more versatile depending on the strain. This is the first study pointing out and comparing different metabolite patterns in Italian samples of Pleurotus species, including P. eryngii varieties.

Fomitopsis officinalis: Spatial (Pileus and Hymenophore) Metabolomic Variations Affect Functional Components and Biological Activities.

Fomitopsis officinalis is a holartic polyporous mushroom that forms large fruiting bodies on old standing trees, fallen logs, or stumps. F. officinalis is a medicinal mushroom species that is most commonly used in traditional European medicine. In this study, we explore the spatial metabolic differences in F. officinalis' mushroom parts, i.e., the cap (median and apical parts) and the hymenium. Additionally, chromatographic analysis was conducted in order to unravel the composition of specialized metabolites in the hydroalcoholic mushroom extracts. The potential antifungal and bacterial effects of extracts were tested against pathogen strains of Gram+ and Gram- bacteria, and yeast, dermatophytic, and fungal-pool species. Extracts from the apical part were the richest in terms of phenolic compounds; consistent with this finding, the extracts were also the most effective antiradical and antimicrobial agents with MIC values < 100 µg/mL for most of the tested bacterial and dermatophytic species. According to these findings, F. officinalis extracts are valuable sources of primary and secondary metabolites, thus suggesting potential applications in the formulation of food supplements with biological properties in terms of antioxidant and antimicrobial activities.

Unconventional Extraction of Total Non-Polar Carotenoids from Pumpkin Pulp and Their Nanoencapsulation.

Pumpkin is considered a functional food with beneficial effects on human health due to the presence of interesting bioactives. In this research, the impact of unconventional ultrasound-assisted extraction (UAE) and microwave-assisted extraction techniques on the recovery of total non-polar carotenoids from Cucurbita moschata pulp was investigated. A binary (hexane:isopropanol, 60:40 v/v) and a ternary (hexane:acetone:ethanol, 50:25:25 v/v/v) mixture were tested. The extracts were characterized for their antioxidant properties by in vitro assays, while the carotenoid profiling was determined by high-performance liquid chromatography coupled with a diode array detector. UAE with the binary mixture (30 min, 45 °C) was the most successful extracting technique, taking into consideration all analytical data and their correlations. In parallel, solid lipid nanoparticles (SLN) were optimized for the encapsulation of the extract, using ß-carotene as a reference compound. SLN, loaded with up to 1% ß-carotene, had dimensions (~350 nm) compatible with increased intestinal absorption. Additionally, the ABTS ((2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assay showed that the technological process did not change the antioxidant capacity of ß-carotene. These SLN will be used to load an even higher percentage of the extract without affecting their dimensions due to its liquid nature and higher miscibility with the lipid with respect to the solid ß-carotene.

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.

Lipidomic profiling of Pleurotus ostreatus by LC/MS Q-TOF analysis.

In this paper, for the first time a lipidomic analysis on Pleurotus ostreatus species was performed by liquid chromatography quadrupole time-of-flight mass spectrometry (LC/MS Q-TOF). Twenty-seven lipid classes, including polar and non-polar lipid classes, were detected. Free fatty acids (FA) were the predominant fraction (>57%), followed by fatty acid ester of hydroxyl fatty acid and ceramide. C18 chain length and two double bonds were the main structural characteristics for FA. Phosphatydilcholine, phosphatydiletanolamine, and glycerophosphates showed high percentages of polyunsaturated fatty acids. Unconventional fatty acids, such as odd and oxygenated chains, were detected. The highest odd/even ratio was found in hexosylceramides and sphingomyelin, while oxygenated chains were mainly represented in ceramides. As a preliminary approach, the results of lipid molecular species, subjected to principal component analysis and discriminant analysis, were able to differentiate P. ostreatus samples on the base of grown substrate. The results of the comprehensive analysis of P. ostreatus lipids are useful to evaluate the lipid nutritional value and could facilitate exploitation of P. ostreatus consumption.

In-depth characterization of phenolic profiling of Moraiolo extra-virgin olive oil extract and initial investigation of the inhibitory effect on Indoleamine-2,3-Dioxygenase (IDO1) enzyme.

In this research, the phenolic extract from Moraiolo extra-virgin olive oil (EVOO) was thoroughly characterized. A reversed-phase HPLC method with a photodiode detector allowed to measure a total phenol content higher than 500 mg/kg EVOO, with elevated amounts of oleocanthal, oleacein, and oleouropein aglycone (131.2, 213.5, and 158.4 mg/kg EVOO, respectively). Appreciable amounts of (+)-pinoresinol and (+)- 1-acetoxy-pinoresinol, 3.2 and 12.5 mg/kg EVOO respectively, were measured. High-resolution mass spectrometry with Orbitrap mass analyzer technology was used to confirm the identity of the analytes. Afterwards, the extract was tested, for the first time, for its activity on Indoleamine-2,3-Dioxygenase (IDO1). This enzyme appears as a promising target for the modulation of the neuroinflammatory-oxidative processes relying on the pathogenesis of several neurodegenerative diseases. The extract showed an inhibitory effect on the catalytic activity of both human and murine IDO1, with a good safety at the concentrations of 15 and 30 µg/mL.

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.

Preparation and characterization of polymeric microparticles loaded with Moringa oleifera leaf extract for exuding wound treatment.

The aim of this study was to develop an innovative formulation, particularly useful for the treatment of exuding wounds. An extract from Moringa oleifera leaves (MOE), prepared by an eco-friendly method, was used as active ingredient. Its preliminary characterization showed that MOE is rich in quercetin-O-glucoside and quercetin-O-malonyl glucoside, responsible for the antioxidant, radical scavenging and antibacterial activities (toward Staphylococcus aureus, S. epidermidis, S. faecalis and S. pyogenes). Moreover, MOE showed the ability to stimulate keratinocytes growth. Thus, bioadhesive biocompatible polymeric microparticles loaded with such extract were developed and prepared in order to treat exuding wounds. The microparticles, obtained by spray drying, using chitosan as polymer, showed good swelling ability. This is useful to obtain the transition from microparticles to a continuous gel covering the wound, after deposition on it. This has the double function to protect the damage area and to promote the healing. The in vitro release study showed that the formed gel is able to release immediately MOE, in the first minutes after application, and to promote a sustained release within 24 h reaching an efficacious concentration against the most sensitive bacterial strains. These findings suggest that the developed microparticles represent an interesting tool for exuding wounds treatment.

Changes of milk fatty acid composition in four lipid classes as biomarkers for the diagnosis of bovine ketosis using bioanalytical Thin Layer Chromatography and Gas Chromatographic techniques (TLC-GC).

The aim of this study was to extend the limited research available on the association between the concentration of milk fatty acids and the elevated plasmatic value of ß-hydroxybutyrate (BHB) in early lactation of dairy cows. Fifty-four Holstein Friesian dairy cows were enrolled in the study. All animals were classified on the basis of their blood BHB concentration: BHB ≥ 1.0 mmol/L (BHB-1, sick group) and BHB ≤ 0.99 mmol/L (BHB-0, healthy group). Using Thin Layer Chromatography (TLC), four lipid classes (cholesterol esters -CE-, phospholipids -PL-, free fatty acids -FFA- and triacylglycerols -TAG-) were separated, and then the fatty acid (FA) composition was determined by High Resolution Gas Chromatography coupled with Flame Ionization Detector/Mass Spectrometer (HRGC-FID/MS). The FA profiles were used to investigate the diagnostic potential value of milk fatty acids for the correct classification of cows with BHB concentration above the established threshold (BHB < 1.0 mmol/L). Boruta Test and Receiver Operating Characteristic curves (ROC) were used to identify which FA and their thresholds of concentration could be used when animals presented hyperketonemia. The research has identified fourteen FA, belonging to CE, FFA, and TAG classes, useful for an association with BHB-1. These compounds, with predictive value for the development of hyperketonemia, could be considered valuable biomarkers. Further studies on a wider sampling, based on clinical and therapeutic approach, will be necessary to confirm, by bioanalytical chromatographic approaches, if these predictive FA will change between healthy and sick animals. New approaches in relation on the administration of different diets or supplements, and administration of drugs might improve the prevention of hyperketonemia.

Phenolic profiling and in vitro bioactivity of Moringa oleifera leaves as affected by different extraction solvents.

In this work the (poly)-phenolic profile of Moringa oleifera leaves was comprehensively investigated through untargeted metabolomics, following a homogenizer-assisted extraction (HAE) using three solvent systems, i.e. methanol (HAE-1), methanol-water 50:50 v/v (HAE-2) and ethyl acetate (HAE-3). This approach allowed to putatively annotate 291 compounds, recording mainly flavonoids and phenolic acids. Thereafter, antioxidant capacity, antimicrobial activity and enzyme inhibition were assayed in the different extracts. HAE-1 extract showed the highest total phenolic content (31.84 mg/g), followed by HAE-2 (26.95 mg/g) and HAE-3 (14.71 mg/g). In addition, HAE-1 and HAE-2 extracts exhibited an expressive activity against Bacillus cereus and Listeria innocua. The HAE-2 leaf extract was characterized by the highest DPPH and ABTS values (being 49.55 and 45.26 mgTE/g), while ferric reducing antioxidant power was found to be higher in HAE-1 (58.26 mgTE/g). Finally, the enzyme inhibitory effects of M. oleifera leaf extracts were investigated against five enzymes, namely acetylcholinesterase (AChE), butyrylcholinesterase (BChE), tyrosinase, α-amylase and α-glucosidase. All of the tested extracts exhibited inhibitory effects on AChE and BChE with a higher activity for HAE-3 and HAE-1, whilst HAE-1 showed the higher impact on tyrosinase, glucosidase and amylase activities. Taken together, these findings suggest that M. oleifera leaf extracts are a good source of bioactive polyphenols with a potential use in food and pharma industries.

Artocarpus tonkinensis Protects Mice Against Collagen-Induced Arthritis and Decreases Th17 Cell Function.

Artocarpus tonkinensis (Moraceae) is a tree that grows in north Vietnam whose leaf decoction is used as a traditional remedy by the Hmong ethnic group to treat arthritis and backache. Our study evaluated the decoction's efficacy and mechanism of action in DBA/1J mice with collagen-induced arthritis (CIA). Mice treated with the decoction (At) either from the first collagen immunization or after CIA development experienced significantly less joint edema and inflammatory infiltration, whereas CIA-induced cartilage damage could only be prevented by early At treatment. Autoimmune gene expression profiles showed that Th17 cell-associated chemokine CCL20 and cytokines IL-6, IL-17, and IL-22 were strongly downregulated by At. Reduced expression of IL-2, IL-17, IL-22, and FasL in lymph node cells from At-treated mice was further confirmed by real-time PCR. The decoction also inhibited polarization of Th17 cells from CD4+ splenic T cells according to levels of IL-17 and RORC, a Th17 cell-specific transcription factor. Chromatographic analysis identified At's major component as maesopsin-ß-D-glucoside, which could inhibit in vitro differentiation of Th17 cells. The decoction significantly alleviated the signs and symptoms of CIA and inhibited the development and function of Th17 cells, highlighting its potent anti-inflammatory activity.

Impact of conventional/non-conventional extraction methods on the untargeted phenolic profile of Moringa oleifera leaves.

The impact of different extraction methods, namely maceration, homogenizer-assisted extraction, rapid solid-liquid dynamic extraction, microwave-assisted extraction and ultrasound-assisted extraction, on polyphenols of Moringa oleifera leaves was studied. The phenolic composition of alcoholic (methanol 100%) and hydroalcoholic (methanol/water 50:50, v/v) extracts was compared by using an untargeted metabolomics-based profiling approach followed by multivariate statistics. With this aim, ultra-high-pressure liquid chromatography coupled to a quadrupole-time-of-flight mass spectrometry was used to profile phenolic compounds under the different extraction conditions. Besides, the in vitro antioxidant activities of Moringa leaves were also investigated as ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC). The metabolomic approach allowed to putatively annotate 262 phenolic compounds. In particular, glycosidic forms of quercetin (i.e., quercetin 3-O-galactoside, quercetin 3-O-glucoside, and quercetin 4'-O-glucoside) were the most represented compounds among flavonoids. Furthermore, protocatechuic acid was found to be the most abundant hydroxybenzaldheyde derivative, while the isomeric forms of hydroxybenzoic acid characterized the phenolic acids class. Overall, the extractions in methanol 100% were found to be the most effective for phenolic compounds recovering, when compared with those in methanol/water (50:50, v/v). Homogenizer-assisted extraction of M. oleifera leaves using 100% methanol allowed extracting the highest amounts of polyphenols (35.19 mg/g) and produced the highest oxygen radical absorbance capacity (536.27 µmol Trolox Equivalents/g). The supervised orthogonal projection to latent structures discriminant analysis identified phenolic acids as the phenolic class mostly affected by the different extraction technologies. These findings demonstrate that each extraction method promoted the recovery of specific phenolic subclasses with different efficiencies.

In Vitro Safety/Protection Assessment of Resveratrol and Pterostilbene in a Human Hepatoma Cell Line (HepG2).

The aim of this work was to evaluate in vitro the genotoxic and/or antigenotoxic effects of resveratrol (RESV) and pterostilbene (PTER) on HepG2 cells. Moreover, additional tests were performed to evaluate early and late apoptosis events induced by the tested stilbenes. RESV and PTER did not show any genotoxic activity. As regards antigenotoxicity testing, RESV and PTER showed a typical, U-shaped hormetic dose-response relationship characterized by a biphasic trend with small quantities having opposite effects to large ones. HepG2 cells treated with PTER exhibited a marked increase in early apoptosis (40.1%) at 250 microM; whereas, the highest concentration tested for both RESV and PTER significantly increased the proportion of HepG2 cells undergoing late apoptosis (32.5 and 51.2%, respectively). The observed pro-apoptotic activity could, at least in part, explain the hormetic response observed when the compounds were tested for antigenotoxicity (i.e., in the presence of induced DNA damage).

Characterisation of secondary metabolites in saffron from central Italy (Cascia, Umbria).

Saffron's quality depends on the concentration of secondary metabolites, such as crocins, picrocrocin and safranal. The aim of this research was to evaluate the influence of drying conditions on the secondary metabolite contents of saffron produced in the area of Cascia, in central Italy. Different aliquots of the same saffron sample were subjected to various dehydration conditions and analysed by UV-Vis spectrophotometry to determine crocins, picrocrocin and safranal.. Safranal was also analysed by high resolution gas chromatography, while the crocins and picrocrocin were determined by high-performance liquid chromatography with diode array and mass spectrometric detectors. The results of chromatographic analyses showed that the samples dried in the milder conditions had the lowest content of secondary metabolites. Moreover the sample dried at 60°C for 55min presented the highest contents of trans-crocin-4 and picrocrocin, while safranal was most represented in saffron dried at 55°C for 95min.

Original normal-phase high-performance liquid chromatographic separation of monoacylglycerol classes from extra virgin olive oil triacylglycerols for their stereospecific analysis.

An innovative procedure to separate the 3 isomeric sn-monoacylglycerols (MAG) classes (sn-1-, sn-2-, sn-3-MAG) is described. MAGs, obtained by chemical deacylation of triacylglycerols (TAGs), have been derivatized with (S)-(+)-1-(1-naphtyl)ethyl-isocyanate, and the resulting urethane derivatives have been separated by normal-phase high-performance liquid chromatography. This procedure allows resolution as diasteroisomers of the 2 enantiomeric classes (sn-1-MAG and sn-3-MAG), without the need of a chiral column, and to separate also the isomeric sn-2-MAG class; moreover, by introducing a chromophoric moiety, this reagent makes possible the ultraviolet detection of the analyte molecules. This procedure has been used to obtain the stereospecific analysis of the TAG fraction of extra virgin olive oil samples. The use of a nondestructive detector permitted the collection of the individual urethane classes; the fatty acid composition of each was determined by high-resolution gas chromatography, obtaining directly from the data the fatty acid distribution within each sn- position of TAGs. To validate this new method, the results have been compared with those obtained by 2 other procedures for TAG stereospecific analysis, and the obtained results were satisfactory since the proposed method gave data very similar to the other procedures.