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Compounds present in Cannabis sativa L. preparations have recently attracted much attention in the treatment of drug-resistant epilepsy. Here, we screened two olive oil extracts from a non-psychoactive C. sativa variety, fully characterized by high-performance liquid chromatography and gas chromatography. Particularly, hemp oils with different concentrations of terpenes were administered at the same dose of cannabidiol (25 mg/kg/day orally), 1 h before the 6-Hz corneal stimulation test (44 mA). Mice were stimulated once a day for 5 days and evaluated by video-electrocorticographic recordings and behavioral analysis. Neuronal activation was assessed by FosB/ΔFosB immunoreactivity. Both oils significantly reduced the percentage of mice experiencing convulsive seizures in comparison to olive oil-treated mice ( p < 0.050; Fisher's exact test), but only the oil enriched with terpenes (K2) significantly accelerated full recovery from the seizure. These effects occurred in the presence of reduced power of delta rhythm, and, instead, increased power of theta rhythm, along with a lower FosB/ΔFosB expression in the subiculum ( p < 0.050; Duncan's method). The overall findings suggest that both cannabinoids and terpenes in oil extracts should be considered as potential therapeutic agents against epileptic seizures and epilepsy.

In this study, Thymus numidicus Poir. plant material was collected from two different locations in north-western Tunisia and the aerial parts essential oils (EOs) were extracted via hydro-distillation. Gas chromatography coupled to mass spectrometry (GC-MS) and flame ionisation detection (GC-FID) were used for the qualitative analysis and quantification of the volatile constituents. Thymol (50.1-52.8%) was identified as the main compound of both EOs. To evaluate the potential application of the EOs as antifungal agents, the in vitro inhibitory effects were tested against six fungal strains; a strong antifungal activity of one sample was observed (MIC = 40-400 µg/mL). The in vitro antiproliferative activity was investigated on two human cancer cell lines, i.e. the colonic (HCT116) and breast adenocarcinoma (MCF7) using the colourimetric MTT assay. Again, the same sample demonstrated to possess good antiproliferative activity against both cancer cell lines, with IC 50 values of 26.9 and 11.7 µg/mL, respectively.

Cannabis sativa L. is a plant known all over the world, due to its history, bioactivity and also social impact. It is chemically complex with an astonishing ability in the biosynthesis of many secondary metabolites belonging to different chemical classes. Among them, cannabinoids are the most investigated ones, given their pharmacological relevance. In order to monitor the composition of the plant material and ensure the efficacy and safety of its derived products, extraction and analysis of cannabinoids play a crucial role. In this context, in addition to a conventional separation method based on HPLC with UV/DAD detection, a new strategy based on a non-separation procedure, such as 13 C-qNMR, may offer several advantages, such as reduced solvent consumption and simultaneous acquisition of the quali/quantitative data related to many analytes. In the light of all the above, the aim of this work is to compare the efficiency of the above-mentioned analytical techniques for the study of the main cannabinoids in different samples of cannabis inflorescences, belonging to fibre-type, recreational and medical varieties. The 13 C-qNMR method here proposed for the first time for the quantification of both psychoactive and non-psychoactive cannabinoids in different cannabis varieties provided reliable results in comparison to the more common and consolidated HPLC technique., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In this study, the phenol loading and antioxidant activity of wool yarn prepared with the aqueous extract of onion ( Allium cepa L.) skin was enhanced by implementing the dyeing process with the use of alum as a mordant. Spectrophotometric and chromatographic methods were applied for the characterization of polyphenolic substances loaded on the wool yarn. The antioxidant/anti-inflammatory properties were evaluated by determining the level of intra- and extra-cellular reactive oxygen species (ROS) production in keratinocytes and dermal fibroblasts pre-treated with lipopolysaccharide put in contact with artificial sweat. An elevated dye uptake on wool was observed for the pre-mordanted sample, as demonstrated by high absorbance values in the UV-Visible spectral range. Chromatographic results showed that protocatechuic acid and its glucoside were the main phenolic acid released in artificial sweat by the wool yarns, while quercetin-4'-glucoside and its aglycone quercetin were more retained. The extract released from the textile immersed in artificial sweat showed a significant reducing effect on the intra-and extracellular ROS levels in the two cell lines considered. Cytofluorimetric analyses demonstrated that the selected mordant was safe at the concentration used in the dyeing procedure. Therefore, alum pre-mordanted textiles dyed with onion-skin extracts may represent an interesting tool against skin diseases.

An original, innovative, high-throughput method based on attenuated total reflectance - Fourier's transform infrared (ATR-FTIR) spectroscopy has been developed for the proof-of-concept discrimination of fibre-type from drug-type Cannabis sativa L. inflorescences. The cannabis sample is placed on the instrument plate and analysed without any previous sample pretreatment step. In this way, a complete analysis lasts just a few seconds, the time needed to record an ATR-FTIR spectrum. The method was calibrated and cross-validated using data provided by liquid chromatography - tandem mass spectrometry (LC-MS/MS) analysis of the different cannabis samples and carried out the statistical assays for quantitation. During cross-validation, complete agreement was obtained between ATR-FTIR and LC-MS/MS identification of the cannabis chemotype. Moreover, the method has proved to be capable of quantifying with excellent accuracy (75-103 % vs. LC-MS/MS) seven neutral and acidic cannabinoids (THC, THCA, CBD, CBDA, CBG, CBGA, CBN) in inflorescences from different sources. The extreme feasibility and speed of execution make this ATR-FTIR method highly attractive as a proof-of-concept for a possible application to quality controls during pharmaceutical product manufacturing, as well as on-the-street cannabis controls and user counselling., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Recently, 1-DNJ has been widely studied by scientists for its capacity to inhibit α-glucosidase and reduce postprandial blood glucose and fat accumulation. To the best of our knowledge, this is the first analytical determination of 1-DNJ in Morus sp.pl. leaves carried out on Italian crops, and it could be used as a reference to assess the quality of the plant material in comparison to Far Eastern Asia cultivations. The effects of two thermal treatments were compared to test the incidence of the drying process on the 1-DNJ extractability. In addition, two harvesting seasons in the same year (2017) and two subsequent harvesting years (2017-2018) were considered. The amount of 1-DNJ herein found was comparable to that reported in the scientific literature for Asian cultivations. The increase in 1-DNJ along the summer and the higher level of this compound in the apical leaves also complies with previous findings. However, a strong implication for the climatic conditions in the different years and a significant interaction between climate and genotypes suggest exploring very carefully the agronomic practices and selecting cultivars according to different environmental conditions with a view to standardize the 1-DNJ amount in leaves.

Introduction: The ever-growing diffusion and consumption of herbal teas, due to their sensory attributes and well-known health benefits exposes them to the real risk of adulteration, especially in the case of commercial mixtures already minced for infusion. Therefore, novel and suitable tools for the control of these valuable products are increasingly required., Objectives: This work provides new insights for the authenticity study of infusions. The main objective was verifying the potential of proton nuclear magnetic resonance ( 1 H-NMR) combined with partial least square (PLS) regression to build highly predictive models, useful for the determination of the real amounts of herbs in mixtures, by the simple analysis of the related infusion., Materials and Methods: Peppermint, fennel, lemon balm, and passiflora were chosen to set-up an experimental plan according to a central composite design (CCD). One-dimensional nuclear Overhauser effect spectroscopy (1D-NOESY) spectra were properly pretreated and then analysed by chemometrics to extract significant information from the raw data., Results: Venetian-blind cross-validation and different chemometric indicators (RMSEC, RMSECV, RMSEP, R 2 CAL , R 2 CV, R 2 PRED ) were used to establish the best model, which include four factors explaining 88.70 and 83.77% of the total variance in X and Y, respectively., Conclusions: These promising results have laid the basis for further development of the method, to extend its applicability and make it more scalable. This tool could replace expensive separative techniques and protect the rights of consumers with particular attention to safety issues and quality assurance., (© 2020 John Wiley & Sons, Ltd.)

Coronavirus Disease 2019 (COVID-19) is upsetting the world and innovative therapeutic solutions are needed in an attempt to counter this new pandemic. Great hope lies in vaccines, but drugs to cure the infected patient are just as necessary. In the most severe forms of the disease, a cytokine storm with neuroinflammation occurs, putting the patient's life at serious risk, with sometimes long-lasting sequelae. Palmitoylethanolamide (PEA) is known to possess anti-inflammatory and neuroprotective properties, which make it an ideal candidate to be assumed in the earliest stage of the disease. Here, we provide a mini-review on the topic, pointing out phospholipids consumption in COVID-19, the possible development of an antiphospholipid syndrome secondary to SARS-CoV-2 infection, and reporting our preliminary single-case experience concerning to a 45-year-old COVID-19 female patient recently treated with success by micronized / ultramicronized PEA., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Punica granatum L. (pomegranate) fruit is known to be an important source of bioactive phenolic compounds belonging to hydrolysable tannins. Pomegranate extracts have shown antifungal activity, but the compounds responsible for this activity and their mechanism/s of action have not been completely elucidated up to now. The aim of the present study was the investigation of the inhibition ability of a selection of pomegranate phenolic compounds (i.e., punicalagin, punicalin, ellagic acid, gallic acid) on both plant and human fungal pathogens. In addition, the biological target of punicalagin was identified here for the first time. The antifungal activity of pomegranate phenolics was evaluated by means of Agar Disk Diffusion Assay and minimum inhibitory concentration (MIC) evaluation. A chemoinformatic analysis predicted for the first time topoisomerases I and II as potential biological targets of punicalagin, and this prediction was confirmed by in vitro inhibition assays. Concerning phytopathogens, all the tested compounds were effective, often similarly to the fungicide imazalil at the label dose. Particularly, punicalagin showed the lowest MIC for Alternaria alternata and Botrytis cinerea , whereas punicalin was the most active compound in terms of growth control extent. As for human pathogens, punicalagin was the most active compound among the tested ones against Candida albicans reference strains, as well as against the clinically isolates. UHPLC coupled with HRMS indicated that C. albicans , similarly to the phytopathogen Coniella granati , is able to hydrolyze both punicalagin and punicalin as a response to the fungal attack. Punicalagin showed a strong inhibitory activity, with IC 50 values of 9.0 and 4.6 µM against C. albicans topoisomerases I and II, respectively. Altogether, the results provide evidence that punicalagin is a valuable candidate to be further exploited as an antifungal agent in particular against human fungal infections.

Cannabidiol (CBD) is a bioactive terpenophenolic compound isolated from Cannabis sativa L. It is known to possess several properties of pharmaceutical interest, such as antioxidant, anti-inflammatory, anti-microbial, neuroprotective and anti-convulsant, being it active as a multi-target compound. From a therapeutic point of view, CBD is most commonly used for seizure disorder in children. CBD is present in both medical and fiber-type C. sativa plants, but, unlike Δ 9 -tetrahydrocannabinol (THC), it is a non-psychoactive compound. Non-psychoactive or fiber-type C. sativa (also known as hemp) differs from the medical one, since it contains only low levels of THC and high levels of CBD and related non-psychoactive cannabinoids. In addition to medical Cannabis, which is used for many different therapeutic purposes, a great expansion of the market of hemp plant material and related products has been observed in recent years, due to its usage in many fields, including food, cosmetics and electronic cigarettes liquids (commonly known as e-liquids). In this view, this work is focused on recent advances on sample preparation strategies and analytical methods for the chemical analysis of CBD and related compounds in both C. sativa plant material, its derived products and biological samples. Since sample preparation is considered to be a crucial step in the development of reliable analytical methods for the determination of natural compounds in complex matrices, different extraction methods are discussed. As regards the analysis of CBD and related compounds, the application of both separation and non-separation methods is discussed in detail. The advantages, disadvantages and applicability of the different methodologies currently available are evaluated. The scientific interest in the development of portable devices for the reliable analysis of CBD in vegetable and biological samples is also highlighted., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Current therapies for neuropathic pain are generally symptomatic and possess several side effects, limiting their prolonged usage., Hypothesis/purpose: Thus, it is urgent to develop novel and safe candidates for the management of this chronical condition. For this purpose, we investigated the analgesic effect of a standardized extract from Zingiber officinale Roscoe rhizomes (ZOE) obtained by CO 2 supercritical extraction, in a mice model of peripheral neuropathy. We also explored the mechanism of action of ZOE and its main constituents using an in vitro model of neuroinflammation., Methods: Peripheral mono-neuropathy was induced in mice, by spared nerve injury (SNI). The analgesic effect of ZOE after oral administration was assessed by measuring mechanical and thermal allodynia in SNI mice. The mechanism of action of ZOE and its main constituents were investigated using spinal cords samples and in an in vitro model of neuroinflammation by ELISA, western blotting and immunofluorescence techniques., Results: Oral administration of ZOE 200 mg kg -1 ameliorated mechanical and thermal allodynia in SNI mice, with a rapid and a long-lasting effect. ZOE did not alter locomotor activity. In BV2 cells and spinal cord samples, ZOE, 6-gingerol and 6-shogaol reduced pERK levels, whereas ZOE and terpene fraction reduced HDAC1 protein levels, inhibited NF-κB signalling activation and decreased IL-1β, TNF-α and IL-6 release. ZOE and each tested constituent had a positive effect on inflammation-impaired SH-SY5Y cell viability., Conclusions: The oral administration of ZOE attenuated SNI-induced neuropathic pain symptoms by reducing spinal neuroinflammation, suggesting ZOE as a novel and interesting candidate for the management of neuropathic pain., Competing Interests: Declaration of Competing Interest The authors declared no conflict of interest., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

To improve the loadability and antioxidant properties of wool impregnated with onion skin extract, the introduction of SB3-14 surfactant in the dyeing process was evaluated. A preliminary investigation on the surfactant-quercetin interaction indicated that the optimal conditions for dye solubility, stability, and surfactant affinity require double-distilled water (pH = 5.5) as a medium and SB3-14 in a concentration above the c.m.c. (2.5 × 10 -3 M). The absorption profile of textiles showed the flavonoid absorption band (390 nm) and a bathochromic feature (510 nm), suggesting flavonoid aggregates. The higher absorbance for the sample dyed with SB3-14 indicated greater dye uptake, which was further confirmed by HPLC analysis. The Folin-Ciocalteu method was applied to evaluate the total phenol content (TPC) released from the treated wool, while the assays FRAP, DPPH, ABTS, and ORAC were applied to evaluate the corresponding total antioxidant activity (TAC). Higher TPCs (about 20%) and TACs (5-55%) were measured with SB3-14, highlighting textiles with improved biofunctional properties. Spectrophotometric analyses were also performed with an artificial sweat. The potential cytotoxic effect of SB3-14 in both monomeric and aggregated forms, cell viability, and induction of apoptosis were evaluated in RAW 264.7 cells. These analyses revealed that SB3-14 is safe at concentrations below the c.m.c.

The scientific interest on the plant Cannabis sativa L., and in particular on its non-psychoactive or fibre-type variety (hemp), has been highly increasing in recent years, due to the pharmaceutical and nutraceutical potential of its bioactive compounds. This plant is indeed characterized by a very rich chemical composition, which encompasses different classes of constituents, such as cannabinoids and terpenes. In this context, the bioanalytical testing of hemp extracts can be difficult and time-consuming. Effect-directed analysis (EDA) by the combination of high-performance thin-layer chromatography (HPTLC) with biological and enzymatic assays represents one of the latest tools available for the rapid bioprofiling of complex matrices, such as plant extracts. In this ambit, the aim of this project was the non-targeted screening of inflorescence extracts from ten different hemp varieties for components exhibiting radical scavenging, antibacterial, enzyme inhibiting and estrogen-like effects. By HPTLC-EDA, the hemp samples exhibited strong antibacterial activities against both Gram-positive Bacillus subtilis and especially Gram-negative Aliivibrio fischeri bacteria, and also estrogen-like activity. They also inhibited α- and β-glucosidase, tyrosinase and acetylcholinesterase. The characterization of two prominently multipotent bioactive compound zones was finally achieved by HPTLC-HRMS and preliminary assigned as cannabidiolic acid and cannabidivarinic acid., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The antimicrobial activity of different essential oils (EOs) from the Lamiaceae family was evaluated on Streptococcus agalactiae , Candida albicans , and lactobacilli. S. agalactiae is the main cause of severe neonatal infections, such as sepsis, meningitis, and pneumonia. C. albicans is a primary causative agent of vulvovaginal candidiasis, a multifactorial infectious disease of the lower female reproductive tract. Lactobacilli represent the dominant bacterial species of the vaginal flora and constitute the natural defense against pathogens. On the basis of the preliminary results, the attention was focused on the EOs from Lavandula x intermedia Emeric ex Loisel. and Mentha arvensis L. By using gas ghromatography (GS) retention data and mass spectra, it was possible to identify more than 90% of the total composition of the EO samples. The minimal inhibitory concentration (MIC) and anti-biofilm activity of the two EOs were determined against all isolated strains, using the EOs by themselves or in combination with each other and with drugs (erythromycin and fluconazole). The results showed a good antimicrobial and anti-biofilm activity of both EOs and a synergistic effect, leading to the best results against all the strains, resulted using the combinations EOs/EOs and antimicrobials/EOs.

Cannabis-based medications are being increasingly used for the treatment of different clinical conditions. Among all galenic formulations, olive oil extracts from medical Cannabis are the most prescribed ones for their easy preparation and usage. A great variety of methods have been described so far for the extraction of medical Cannabis oils to reach a high yield of Δ 9 -tetrahydrocannabinol (Δ 9 -THC), but poor attention has been paid to the preservation of the terpene fraction from the plant, which may contribute to the overall bioactivity of the extracts. In this context, the present study was aimed at the chemical characterization of different medical Cannabis oils prepared by following both innovative and existing extraction protocols, with particular attention to cannabinoids and terpenes, in order to set up a suitable method to obtain an extract rich in these chemical classes. In particular, six different extraction procedures were followed, based on different techniques, of which all but one included a decarboxylation of the plant material. The profile of cannabinoids was studied in detail by means of HPLC-ESI-MS/MS, while terpenes were characterized by means both GC-MS and GC-FID techniques coupled with solid-phase microextraction operated in the head-space mode (HS-SPME). An innovative method that is based on the extraction of the oil by dynamic maceration at room temperature from plant inflorescences, which were partially decarboxylated in a closed system at a moderate temperature and partially pre-extracted with ethanol, produced similar yields of bioactive compounds as that obtained by using a microwave-assisted distillation of the essential oil from the plant material, in combination with a maceration extraction of the oil from the residue. Both these new methods provided a higher efficiency over already existing extraction procedures of medical Cannabis oils and they can be applied to obtain a product with a high therapeutic value., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Anthocyanins have been associated with several health benefits, although the responsible mechanisms are not well established yet. In the present study, an anthocyanin-rich extract from bilberry ( Vaccinium myrtillus L.) was tested in order to evaluate its capacity to modulate reactive oxygen species (ROS) production and resistance to thermally induced oxidative stress, using the nematode Caenorhabditis elegans as an in vivo model. The assays were carried out with the wild-type N2 strain and the mutant strains daf-16(mu86) I and hsf-1(sy441) , which were grown in the presence of two anthocyanin extract concentrations (5 and 10 μg/mL in the culture medium) and further subjected to thermal stress. The treatment with the anthocyanin extract at 5 μg/mL showed protective effects on the accumulation of ROS and increased thermal resistance in C. elegans , both in stressed and non-stressed young and aged worms. However, detrimental effects were observed in nematodes treated with 10 μg/mL, leading to a higher worm mortality rate compared to controls, which was interpreted as a hormetic response. These findings suggested that the effects of the bilberry extract on C. elegans might not rely on its direct antioxidant capacity, but other mechanisms could also be involved. Additional assays were performed in two mutant strains with loss-of-function for DAF-16 (abnormal DAuer Formation factor 16) and HSF-1 (Heat Shock Factor 1) transcription factors, which act downstream of the insulin/insulin like growth factor-1 (IGF-1) signaling pathway. The results indicated that the modulation of these factors could be behind the improvement in the resistance against thermal stress produced by bilberry anthocyanins in young individuals, whereas they do not totally explain the effects produced in worms in the post-reproductive development stage. Further experiments are needed to continue uncovering the mechanisms behind the biological effects of anthocyanins in living organisms, as well as to establish whether they fall within the hormesis concept.

The medicinal plant Spathodea campanulata P. Beauv. (Bignoniaceae) has been traditionally applied for the prevention and treatment of diseases of the kidney and urinary system, the skin, the gastrointestinal tract, and inflammation in general. The present work shows for the first time how chemical components from this plant inhibit Helicobacter pylori growth by urease inhibition and modulation of virulence factors. The crude extract and the main fractions of S. campanulata bark were tested on H. pylori isolated strains and the active ones were further fractionated. Fractions and sub-fractions of the plant crude extract were characterized by ultra-high-performance liquid chromatographic tandem high resolution-mass spectrometry detection (UHPLC-HRMS). Several phenolics and triterpenoids were identified. Among the sub-fractions obtained, SB2 showed the capacity to inhibit H. pylori urease in a heterologous bacterial model. One additional sub-fraction (SE3) was able to simultaneously modulate the expression of two adhesins (HopZ and BabA) and one cytotoxin (CagA). The flavonol kaempferol was identified as the most interesting compound that deserves further investigation as a new hit for its capacity to modulate H. pylori virulence factors.

Dystrophin deficiency is associated with alterations in cell physiology. The functional consequences of dystrophin deficiency are particularly severe for muscle physiology, as observed in Duchenne muscle dystrophy (DMD). DMD is caused by the absence of a 427 kDa isoform of dystrophin. However, in addition to muscular dystrophy symptoms, DMD is frequently associated with memory and attention deficits and epilepsy. While this may be associated with a role for dystrophin in neuronal physiology, it is not clear what neuronal alterations are linked with DMD. Our work shows that CA1 pyramidal neurons from DBA/2J-mdx mice have increased afterhyperpolarization compared to WT controls. All the other electrotonic and electrogenic membrane properties were unaffected by this genotype. Finally, basal synaptic transmission, short-term and long-term synaptic plasticity at Schaffer collateral to CA1 glutamatergic synapses were unchanged between mdx and WT controls. These data show that the excitatory component of hippocampal activity is largely preserved in DBA/2J-mdx mice. Further studies, extending the investigation to the inhibitory GABAergic function, may provide a more complete picture of the functional, network alterations underlying impaired cognition in DMD. In addition, the investigation of changes in neuronal single conductance biophysical properties associated with this genotype, is required to identify the functional alterations associated with dystrophin deficiency and clarify its role in neuronal function., Competing Interests: Declaration of competing interest No competing interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Pseudomonas aeruginosa ( P. aeruginosa ) is an opportunistic pathogen responsible for a wide range of clinical conditions, from mild infections to life-threatening nosocomial biofilm-associated diseases, which are particularly severe in susceptible individuals. The aim of this in vitro study was to assess the effects of an Albanian propolis on several virulence-related factors of P. aeruginosa , such as growth ability, biofilm formation, extracellular DNA (eDNA) release and phenazine production. To this end, propolis was processed using three different solvents and the extracted polyphenolic compounds were identified by means of high performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS) analysis. As assessed by a bioluminescence-based assay, among the three propolis extracts, the ethanol (EtOH) extract was the most effective in inhibiting both microbial growth and biofilm formation, followed by propylene glycol (PG) and polyethylene glycol 400 (PEG 400) propolis extracts. Furthermore, Pseudomonas exposure to propolis EtOH extract caused a decrease in eDNA release and phenazine production. Finally, caffeic acid phenethyl ester (CAPE) and quercetin decreased upon propolis EtOH extract exposure to bacteria. Overall, our data add new insights on the anti-microbial properties of a natural compound, such as propolis against P. aeruginosa . The potential implications of these findings will be discussed.

Pilidiella granati , also known as Coniella granati , is the etiological agent of pomegranate fruit dry rot. This fungal pathogen is also well-known as responsible for both plant collar rot and leaf spot. Because of its aggressiveness and the worldwide diffusion of pomegranate crops, the selection of cultivars less susceptible to this pathogen might represent an interesting preventive control measure. In the present investigation, the role of polyphenols in the susceptibility to P. granati of the two royalties-free pomegranate cultivars Wonderful and Mollar de Elche was compared. Pomegranate fruit were artificially inoculated and lesion diameters were monitored. Furthermore, pathogen DNA was quantified at 12-72 h post-inoculation within fruit rind by a real time PCR assay setup herein, and host total RNA was used in expression assays of genes involved in host-pathogen interaction. Similarly, protein extracts were employed to assess the specific activity of enzymes implicated in defense mechanisms. Pomegranate phenolic compounds were evaluated by HPLC-ESI-MS and MS 2 . All these data highlighted 'Wonderful' as less susceptible to P. granati than 'Mollar de Elche'. In the first cultivar, the fungal growth seemed controlled by the activation of the phenylpropanoid pathway, the production of ROS, and the alteration of fungal cell wall. Furthermore, antifungal compounds seemed to accumulate in 'Wonderful' fruit following inoculation. These data suggest that pomegranate polyphenols have a protective effect against P. granati infection and their content might represent a relevant parameter in the selection of the most suitable cultivars to reduce the economic losses caused by this pathogen.

The antibacterial activity of Rosmarinus officinalis L. and Thymus vulgaris L. essential oils (EOs), and their combination against food-borne and spoilage bacteria ( Listeria monocytogenes , Salmonella enteritidis , Yersinia enterocolitica , Escherichia coli and Pseudomonas spp.) was determined. The EOs inhibitory effect was evaluated both in vitro by using the disk diffusion assay and the minimum inhibitory concentration (MIC) determination, and on food by using an artificially contaminated ready-to-eat (RTE) vegetables. The results showed that the lowest MIC values were obtained with R. officinalis and T. vulgaris EOs against E. coli (4 and 8 μL/mL, respectively). The incorporation of the EOs alone or their combination in RTE vegetables reduced the viable counts of all the tested strains. Lastly, in the on food study we simulated the worst hygienic conditions, obtaining results that can be considered a warranty of safety.

Four cultivars (cv) of Lavandula x intermedia ('Abrialis', 'Alba', 'Rinaldi Ceroni' (R.C.) and 'Sumiens') were cultivated in Italy and their essential oils (EOs) were distilled from Alfalfa Mosaic Virus-free plants. These EOs and one from L. angustifolia Miller were chemically characterised by GC-MS and GC-FID. Antimicrobial activity was evaluated against Listeria monocytogenes (24 strains) and Salmonella enterica (10 food strains). Minimal inhibitory concentrations (MIC) ≥ 10.0 μL/mL inhibited Salmonella (cv 'R.C.' was the most active); MIC of 0.3 μL/mL for cv 'Abrialis' and 'R.C.' inhibited L. monocytogenes , revealing noticeable activity, especially on clinical strains. This activity appears related to EOs composition. Particularly cv 'Abrialis' and 'R.C.' showing the highest antimicrobial activity, were rich in the specific constituents: linalool (38.17 and 61.98%), camphor (8.97 and 10.30%), 1,8-cineole (6.89 and 8.11%, respectively). These EOs could find potential applications in food biopreservation and in surface decontamination, even in hospitals, and deserve deeper investigations.

Policosanols (PCs) are a mixture of long chain primary aliphatic alcohols mainly known for their ability to reduce cholesterol level. Due to this property, there is an increasing interest in the extraction process of these compounds. In this context, beeswax, a natural product produced by honey bees of the genus Apis, is a promising source for their extraction and purification. The present research work was aimed at the development of a new procedure for the extraction and purification of PCs from yellow beeswax by using microwave-assisted technology, which hitherto has never been applied to this mixture. The developed process comprises three main steps: 1) microwave-assisted trans-esterification; 2) microwave-assisted hydrolysis; 3) final purification by means of preparative liquid chromatography. The final step is responsible for the increased purity of PCs, thanks to the removal of undesired compounds, such as natural paraffins. The predominant alcohols investigated in this work are tetracosanol (C 24 OH), hexacosanol (C 26 OH), octacosanol (C 28 OH), triacontanol (C 30 OH) and dotriacontanol (C 32 OH). Compound identification was performed using GC-EI-MS, while GC-FID analysis was chosen for the quantification of the main fatty alcohols present in the product. This new method represents a useful tool for the production of PCs from beeswax to be used in pharmaceuticals and nutraceuticals for human use, feed and veterinary supplements., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Solanum melongena L., also known as eggplant, is a widely consumed vegetable and it is well-known for its beneficial antioxidant properties, due to phenolic compounds. In this work, the influence of different cooking procedures on the content of chlorogenic acid was evaluated on eggplant samples of different geographic origin by high-performance liquid chromatography (HPLC). An easy and quick extraction procedure with 50% methanol as the extraction solvent was optimized for the first time by means of a design-of-experiment and applied to heat treated samples of eggplant. The antioxidant capacity of eggplant extracts was also evaluated by using the ABTS assay and it was correlated with the data obtained by the HPLC method. The content of chlorogenic acid was different in each heat-treated eggplant sample and it depended on the temperature applied during the cooking procedure. In particular, an increase of chlorogenic acid content with rising temperature was observed. Conversely, a very high temperature (250 °C) caused a decrease of chlorogenic acid amount. The influence of storage on the content of chlorogenic acid was also monitored. While the level of chlorogenic acid in fresh samples decreased during four weeks of storage, an increase in its content in heat treated eggplant was observed within the same period. Multivariate data analysis was used to classify eggplant samples into different groups, according to the country of origin and heat treatment procedure. This study provides new insights to preserve the antioxidant properties of eggplant phenolics during different thermal and storage treatments in order to highlight their health promoting effects.

Fibre-type Cannabis sativa L. (hemp) represents a valuable resource in many different fields, including both the pharmaceutical and food ones. This plant contains non-psychoactive cannabinoids, a class of bioactive compounds biosynthesized in both female and male inflorescences. Among them, cannabidiol (CBD) is the most interesting compound from a medicinal point of view. Indeed, several scientific studies have proved its therapeutic potential in a large number of pathologies, in addition to its biological effects attributable to its antioxidant, neuroprotective and anti-inflammatory properties. The analysis of the amount of cannabinoids in food and food supplements represents a critical issue in the ambit of both the quality assurance and the dietary intake control of these biologically active compounds. In this ambit, a particular attention is necessary for apiary products, since they are widely consumed and they can be produced by bees starting from different floral sources. In the light of all the above, the aim of this study was to develop for the first time a new analytical method based on RP-HPLC with ESI-MS/MS detection for the determination of CBD and related cannabinoids in honey. A quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction procedure with an un-buffered method was selected and optimised as the more suitable protocol. As regards detection, it was carried out by using a linear ion trap quadrupole (QTRAP) mass analyser, operated in the multiple reaction monitoring (MRM) mode. Hemp male inflorescences and pollen were analysed in parallel by means of HPLC-UV/DAD, since bees can transfer pollen into their hives and, consequently, into beehive products. The method developed and validated for the first time in this work was finally applied to the analysis of cannabinoids in honey samples, thus demonstrating to be a useful tool for both quality control and safety assurance., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The consumption of high-nutritional-value juice blends is increasing worldwide and, considering the large market volume, fraud and adulteration represent an ongoing problem. Therefore, advanced anti-fraud tools are needed. This study aims to verify the potential of 1 H NMR combined with partial least squares regression (PLS) to determine the relative percentage of pure fruit juices in commercial blends. Apple, orange, pineapple, and pomegranate juices were selected to set up an experimental plan and then mixed in different proportions according to a central composite design (CCD). NOESY (nuclear Overhauser enhancement spectroscopy) experiments that suppress the water signal were used. Considering the high complexity of the spectra, it was necessary to pretreat and then analyze by chemometric tools the large amount of information contained in the raw data. PLS analysis was performed using venetian-blind internal cross-validation, and the model was established using different chemometric indicators (RMSEC, RMSECV, RMSEP, R 2 CAL , R 2 CV , R 2 PRED ). PLS produced the best model, using five factors explaining 94.51 and 88.62% of the total variance in X and Y, respectively. The present work shows the feasibility and advantages of using 1 H NMR spectral data in combination with multivariate analysis to develop and optimize calibration models potentially useful for detecting fruit juice adulteration.

Cannabigerol (CBG) and cannabichromene (CBC) are non-psychoactive cannabinoids that have raised increasing interest in recent years. These compounds exhibit good tolerability and low toxicity, representing promising candidates for drug repositioning. To identify novel potential therapeutic targets for CBG and CBC, an integrated ligand-based and structure-based study was performed. The results of the analysis led to the identification of CBG as a low micromolar inhibitor of the Enoyl acyl carrier protein (ACP) reductase (InhA) enzyme.

Volatile terpenes represent the largest group of Cannabis sativa L. components and they are responsible for its aromatic properties. Even if many studies on C. sativa have been focused on cannabinoids, which are terpenophenolics, little research has been carried out on its volatile terpenic compounds. In the light of all the above, the present work was aimed at the chemical characterization of seventeen essential oils from different fibre-type varieties of C. sativa (industrial hemp or hemp) by means of GC-MS and GC-FID techniques. In total, 71 compounds were identified, and the semi-quantitative analysis revealed that α- and β-pinene, β-myrcene and β-caryophyllene are the major components in all the essential oils analysed. In addition, a GC-MS method was developed here for the first time, and it was applied to quantify cannabinoids in the essential oils. The antibacterial activity of hemp essential oils against some pathogenic and spoilage microorganisms isolated from food and food processing environment was also determined. The inhibitory effects of the essential oils were evaluated by both the agar well diffusion assay and the minimum inhibitory concentration (MIC) evaluation. By using the agar diffusion method and considering the zone of inhibition, it was possible to preliminarily verify the inhibitory activity on most of the examined strains. The results showed a good antibacterial activity of six hemp essential oils against the Gram-positive bacteria, thus suggesting that hemp essential oil can inhibit or reduce bacterial proliferation and it can be a valid support to reduce microorganism contamination, especially in the food processing field.

The aim of this work is to provide a critical review of plant furanocoumarins from different points of view, including their chemistry and biosynthetic pathways to their extraction, analysis, and synthesis, to the main biological activities found for these active compounds, in order to highlight their potential within pharmaceutical science. The limits and the possible improvements needed for research involving these molecules are also highlighted and discussed.

Cannabis sativa L. is a dioecious plant belonging to the Cannabaceae family. The discovery of the presence of many biologically-active metabolites (cannabinoids) in fibre-type Cannabis (hemp) has recently given rise to the valorisation of this variety. In this context, the present study was aimed at the multi-component analysis and determination of the main non-psychoactive cannabinoids (cannabidiol, cannabidiolic acid, cannabigerol and cannabigerolic acid) in female inflorescences of different hemp varieties by means of 13 C quantitative nuclear magnetic resonance spectroscopy (qNMR). The method proposed here for the first time for the determination of cannabinoids provided reliable results in a competitive time with respect to the more consolidated HPLC technique. In fact, it gave sufficiently precise and sensitive results, with LOQ values lower than 750 μg/mL, which is easily achievable with concentrated extracts, without affecting the quality of 13 C-qNMR spectra. In conclusion, this method can be considered as a promising and appropriate tool for the comprehensive chemical analysis of bioactive cannabinoids in hemp and other derived products in order to ensure their quality, efficacy and safety.

Aloysia polystachya (Griseb. et Moldenke) has not been deeply investigated in past years and currently data about its chemical composition are limited. Phenolic compounds characterization can be very difficult in vegetable matrices, owing to bonds to sugar moieties or conjugation, giving rise to complex structures. In this work, methanolic extracts of Aloysia polystachya leaves were analyzed by HPLC-ESI-MS, the favourite technique for the separation and quantification of their polyphenols. To assess the complete characterization and quantification of the phenylpropanoid fraction, three different MS techniques have been coupled to HPLC: ion trap mass spectrometry (Ion Trap LC/MS), quadrupole-time of flight high resolution mass spectrometry (Q-TOF HRMS) and triple-quadrupole (TQ LC/MS) for the quantification. Eleven phenylpropanoid glycosides were identified and quantified and, among them, the compounds forsythoside A, plantainoside C, purpureaside D, martynoside and its two isomers were detected for the first time to the best of our knowledge. The results presented here could be helpful to assess the quality of this plant and could further contribute to the chemotaxonomy of the genus., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The aqueous extract of dry onion skin waste from the 'Dorata di Parma' cultivar was tested as a new source of biomolecules for the production of colored and biofunctional wool yarns, through environmentally friendly dyeing procedures. Specific attention was paid to the antioxidant and UV protection properties of the resulting textiles. On the basis of spectrophotometric and mass spectrometry analyses, the obtained deep red-brown color was assigned to quercetin and its glycoside derivatives. The Folin⁻Ciocalteu method revealed good phenol uptakes on the wool fiber (higher than 27% for the textile after the first dyeing cycle), with respect to the original total content estimated in the water extract (78.50 ± 2.49 mg equivalent gallic acid/g onion skin). The manufactured materials showed remarkable antioxidant activity and ability to protect human skin against lipid peroxidation following UV radiation: 7.65 ± 1.43 (FRAP assay) and 13.60 (ORAC assay) mg equivalent trolox/g textile; lipid peroxidation inhibition up to 89.37%. This photoprotective and antioxidant activity were therefore ascribed to the polyphenol pool contained in the outer dried gold skins of onion. It is worth noting that citofluorimetric analysis demonstrated that the aqueous extract does not have a significative influence on cell viability, neither is capable of inducing a proapoptotic effect.

Cannabis sativa L. represents one of the most widely used source of drugs and drugs of abuse worldwide. Its biologically active compounds are mainly cannabinoids, including Δ 9 -tetrahydrocannabinol (THC), which is responsible for the psychoactive effects, tetrahydrocannabinolic acid (THCA), cannabinol (CBN), cannabidiol (CBD), and cannabidiolic acid (CBDA). Together with recreational and drug-type (or medicinal) Cannabis , some new products have been recently released into the market as fiber-type Cannabis variants (also known as hemp or industrial hemp) with low THC content and high content of nonpsychoactive CBD. In this research work, the aim was to characterize Cannabis recreational and drug-type samples by quantifying their active principles, after the development and validation of a suitable analytical method. In addition to the Cannabis samples described above, fiber-type plant varieties were also analyzed to monitor their content of nonpsychoactive compounds for both pharmaceutical and nutraceutical purposes. To do this, a highly efficient HPLC-DAD-MS/MS method, with an electrospray ionization (ESI) source and a triple-quadrupole mass analyzer acquiring in the multiple reaction monitoring (MRM) mode also coupled to a diode array detector (DAD), was developed and applied. Satisfactory validation results were obtained in terms of precision (RSD < 6.0% for all the analytes) and accuracy (>92.1% for all the compounds). The proposed methodology represents a versatile and reliable tool to assess both psychoactive and nonpsychoactive cannabinoid levels in Cannabis samples for a more rational use in both medicinal chemistry and nutraceutics., Competing Interests: The authors declare no competing financial interest.

In the last decades, a lot of attention has been paid to the compounds present in medicinal Cannabis sativa L., such as Δ 9 -tetrahydrocannabinol (Δ 9 -THC) and cannabidiol (CBD), and their effects on inflammation and cancer-related pain. The National Cancer Institute (NCI) currently recognizes medicinal C. sativa as an effective treatment for providing relief in a number of symptoms associated with cancer, including pain, loss of appetite, nausea and vomiting, and anxiety. Several studies have described CBD as a multitarget molecule, acting as an adaptogen, and as a modulator, in different ways, depending on the type and location of disequilibrium both in the brain and in the body, mainly interacting with specific receptor proteins CB 1 and CB 2 . CBD is present in both medicinal and fibre-type C. sativa plants, but, unlike Δ 9 -THC, it is completely nonpsychoactive. Fibre-type C. sativa (hemp) differs from medicinal C. sativa , since it contains only few levels of Δ 9 -THC and high levels of CBD and related nonpsychoactive compounds. In recent years, a number of preclinical researches have been focused on the role of CBD as an anticancer molecule, suggesting CBD (and CBD-like molecules present in the hemp extract) as a possible candidate for future clinical trials. CBD has been found to possess antioxidant activity in many studies, thus suggesting a possible role in the prevention of both neurodegenerative and cardiovascular diseases. In animal models, CBD has been shown to inhibit the progression of several cancer types. Moreover, it has been found that coadministration of CBD and Δ 9 -THC, followed by radiation therapy, causes an increase of autophagy and apoptosis in cancer cells. In addition, CBD is able to inhibit cell proliferation and to increase apoptosis in different types of cancer models. These activities seem to involve also alternative pathways, such as the interactions with TRPV and GRP55 receptor complexes. Moreover, the finding that the acidic precursor of CBD (cannabidiolic acid, CBDA) is able to inhibit the migration of breast cancer cells and to downregulate the proto-oncogene c-fos and the cyclooxygenase-2 (COX-2) highlights the possibility that CBDA might act on a common pathway of inflammation and cancer mechanisms, which might be responsible for its anticancer activity. In the light of all these findings, in this review we explore the effects and the molecular mechanisms of CBD on inflammation and cancer processes, highlighting also the role of minor cannabinoids and noncannabinoids constituents of Δ 9 -THC deprived hemp.

Echinacea purpurea (L.) Moench, Echinacea angustifolia DC. var. angustifolia and Echinacea pallida (Nutt.) Nutt. are frequently used as medicinal plants and their preparations are among the most widely used herbal medicines. The extracts from these species have shown a highly complex chemical composition, including polar compounds (caffeic acid derivatives, CADs), non-polar compounds (alkylamides and acetylenic secondary metabolites; essential oil) and high molecular weight constituents (polysaccharides and glycoproteins). All these chemical classes of compounds have demonstrated to possess interesting biological activities. In the light of all the above, this paper is focused on the analytical techniques, including sample preparation tools and chromatographic procedures, for the chemical analysis of bioactive compounds in medicinally used Echinacea species. Since sample preparation is considered to be a crucial step in the development of analytical methods for the determination of constituents present in herbal preparations, the strength and weakness of different extraction techniques are discussed. As regards the analysis of compounds present in Echinacea plant material and derivatives, the application of different techniques, mainly HPLC, HPLC-ESI-MS, HPLC-ESI-MS/MS, HPCE, HPTLC and GC, is discussed in detail. The strength, weakness and applicability of the different separation tools are stated., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cannabis sativa L. is a dioecious plant belonging to the Cannabaceae family. The main phytochemicals that are found in this plant are represented by cannabinoids, flavones, and terpenes. Some biological activities of cannabinoids are known to be enhanced by the presence of terpenes and flavonoids in the extracts, due to a synergistic action. In the light of all the above, the present study was aimed at the multi-component analysis of the bioactive compounds present in fibre-type C. sativa (hemp) inflorescences of different varieties by means of innovative HPLC and GC methods. In particular, the profiling of non-psychoactive cannabinoids was carried out by means of HPLC-UV/DAD, ESI-MS, and MS². The content of prenylated flavones in hemp extracts, including cannflavins A and B, was also evaluated by HPLC. The study on Cannabis volatile compounds was performed by developing a new method based on headspace solid-phase microextraction (HS-SPME) coupled with GC-MS and GC-FID. Cannabidiolic acid (CBDA) and cannabidiol (CBD) were found to be the most abundant cannabinoids in the hemp samples analysed, while β-myrcene and β-caryophyllene were the major terpenes. As regards flavonoids, cannflavin A was observed to be the main compound in almost all the samples. The methods developed in this work are suitable for the comprehensive chemical analysis of both hemp plant material and related pharmaceutical or nutraceutical products in order to ensure their quality, efficacy, and safety.

Humulus lupulus L. (hop) represents one of the most cultivated crops, it being a key ingredient in the brewing process. Many health-related properties have been described for hop extracts, making this plant gain more interest in the field of pharmaceutical and nutraceutical research. Among the analytical tools available for the phytochemical characterization of plant extracts, quantitative nuclear magnetic resonance (qNMR) represents a new and powerful technique. In this ambit, the present study was aimed at the development of a new, simple, and efficient qNMR method for the metabolite fingerprinting of bioactive compounds in hop cones, taking advantage of the novel ERETIC 2 tool. To the best of our knowledge, this is the first attempt to apply this method to complex matrices of natural origin, such as hop extracts. The qNMR method set up in this study was applied to the quantification of both prenylflavonoids and bitter acids in eight hop cultivars. The performance of this analytical method was compared with that of HPLC-UV/DAD, which represents the most frequently used technique in the field of natural product analysis. The quantitative data obtained for hop samples by means of the two aforementioned techniques highlighted that the amount of bioactive compounds was slightly higher when qNMR was applied, although the order of magnitude of the values was the same. The accuracy of qNMR was comparable to that of the chromatographic method, thus proving to be a reliable tool for the analysis of these secondary metabolites in hop extracts. Graphical abstract Graphical abstract related to the extraction and analytical methods applied in this work for the analysis of bioactive compounds in Humulus lupulus L. (hop) cones.

Anthocyanins represent the most abundant class of bioactive compounds present in Vaccinium myrtillus L. (bilberry) fruit, conferring it several health-promoting properties. The content of anthocyanins in food products produced from bilberries can be affected by many parameters, making the study of their composition a critical issue. In this ambit, this work was aimed at a comprehensive profiling of anthocyanins in bilberry fruit and derivatives from the Italian Northern Apennines, including jam, juice, and liqueur ("Mirtillino"). Anthocyanins were extracted from the jams by means of a dynamic maceration with acidified methanol, while juice and liqueurs were directly analyzed. The analysis of anthocyanins in the extracts was carried out by means of HPLC-UV/DAD, HPLC-ESI-MS, and MS 2 , under gradient elution. As a comparison, authentic bilberry fruits were analyzed. The total anthocyanin content was in the range 582.4-795.2 mg/100 g (FW) for the fruit, 2.3-234.5 mg/100 g for the jams, 109.2-2252.2 mg/L for the juice, and 27.9-759.3 mg/L for the liqueurs. To deeper investigate the anthocyanin profile of the liqueurs that exhibited a remarkably different composition in comparison with the other products, an authentic bilberry liqueur was prepared in the lab, following a traditional recipe, and monitored weakly by HPLC. The percentage of degradation of 3-O-galactosides and 3-O-arabinosides of bilberry anthocyanidins was found to be higher than that of 3-O-glucosides. The results of this work demonstrated the importance of a suitable and reliable analysis of bilberry fruit and related food products to ensure their genuineness and quality. Graphical abstract Vaccinium myrtillus L. (bilberry) fruit and food products analyzed in this work.

In this study, the activity of essential oils (EOs) against microorganisms involved in oral diseases was evaluated. Fourteen EOs were selected and subjected to gas chromatographic analysis, including Illicium verum, Eucaliptus globulus, Eugenia caryophyllata, Leptospermum scoparium, Mentha arvensis, Mentha piperita, Myrtus communis, Salvia officinalis, Melaleuca alternifolia, Rosmarinus officinalis, Lavandula x intermedia, Thymus capitatus and Thymus vulgaris. These EOs were tested for their antimicrobial activity on Streptococcus mutans and Lactobacillus species clinically isolated from dental surgery patients. The antibacterial activity was evaluated by means of the disc diffusion and the minimum inhibitory concentration (MIC). Five EOs, having shown an interesting antimicrobial activity, were selected for a second screening in combination between them and with chlorhexidine. From the second assays, two EO-EO and three EO-chlorhexidine associations gave interesting results as potential constituents of mouthwashes, especially for the contribution of oxygenated monoterpenes, including menthol, thymol and carvacrol.

The genus Echinacea (Asteraceae) includes species traditionally used in phytotherapy. Among them, Echinacea pallida (Nutt.) Nutt. root extracts are characterized by a representative antiproliferative activity, due to the presence of acetylenic compounds. In this study, supercritical fluid extraction (SFE) was applied and compared with conventional Soxhlet extraction (SE) in order to obtain a bioactive extract highly rich in polyacetylenes and polyenes from E. pallida roots. The composition of the extracts was monitored by means of HPLC-UV/DAD and HPLC-ESI-MS n by using an Ascentis Express C 18 column (150mm×3.0mm I.D., 2.7μm, Supelco, Bellefonte, PA, USA) with a mobile phase composed of (A) water and (B) acetonitrile, under gradient elution. By keeping SFE time at the threshold of 1h (15min static and 45min dynamic for 1 cycle) with the oven temperature set at 40-45°C and 90bar of pressure, an overall extraction yield of 1.18-1.21% (w/w) was obtained, with a high selectivity for not oxidized lipophilic compounds. The biological activity of the extracts was evaluated against human non-small lung A549 and breast carcinoma MCF-7 cancer cell lines. The cytotoxic effect of the SFE extract was more pronounced towards the MCF-7 than the A549 cancer cells, with IC 50 values ranging from 21.01±2.89 to 31.11±2.l4μg/mL; cell viability was affected mainly between 24 and 48h of exposure. The results show the possibility of a new "green" approach to obtain extracts highly rich in genuine polyacetylenes and polyenes from E. pallida roots. The bioactivity evaluation confirmed the cytotoxicity of E. pallida extracts against the considered cancer cell lines, especially against MCF-7 cells, thus suggesting to represent a valuable tool for applicative purposes in cancer prevention., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The present work was aimed at the development and validation of a new, efficient and reliable technique for the analysis of the main non-psychoactive cannabinoids in fibre-type Cannabis sativa L. (hemp) inflorescences belonging to different varieties. This study was designed to identify samples with a high content of bioactive compounds, with a view to underscoring the importance of quality control in derived products as well. Different extraction methods, including dynamic maceration (DM), ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE) and supercritical-fluid extraction (SFE) were applied and compared in order to obtain a high yield of the target analytes from hemp. Dynamic maceration for 45min with ethanol (EtOH) at room temperature proved to be the most suitable technique for the extraction of cannabinoids in hemp samples. The analysis of the target analytes in hemp extracts was carried out by developing a new reversed-phase high-performance liquid chromatography (HPLC) method coupled with diode array (UV/DAD) and electrospray ionization-mass spectrometry (ESI-MS) detection, by using an ion trap mass analyser. An Ascentis Express C 18 column (150mm×3.0mm I.D., 2.7μm) was selected for the HPLC analysis, with a mobile phase composed of 0.1% formic acid in both water and acetonitrile, under gradient elution. The application of the fused-core technology allowed us to obtain a significant improvement of the HPLC performance compared with that of conventional particulate stationary phases, with a shorter analysis time and a remarkable reduction of solvent usage. The analytical method optimized in this study was fully validated to show compliance with international requirements. Furthermore, it was applied to the characterization of nine hemp samples and six hemp-based pharmaceutical products. As such, it was demonstrated to be a very useful tool for the analysis of cannabinoids in both the plant material and its derivatives for pharmaceutical and nutraceutical applications., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Acute respiratory distress syndrome (ARDS) is a serious complication of influenza A (H1N1) virus infection. Its pathogenesis is unknown and biomarkers are lacking. Untargeted metabolomics allows the analysis of the whole metabolome in a biological compartment, identifying patterns associated with specific conditions. We hypothesized that LC-MS could help identify discriminant metabolites able to define the metabolic alterations occurring in patients with influenza A (H1N1) virus infection that developed ARDS. Serum samples from patients diagnosed with 2009 influenza A (H1N1) virus infection with (n = 25) or without (n = 32) ARDS were obtained on the day of hospital admission and analyzed by LC-MS/MS. Metabolite identification was determined by MS/MS analysis and analysis of standards. The specificity of the patterns identified was confirmed in patients without 2009 influenza A(H1N1) virus pneumonia (15 without and 17 with ARDS). Twenty-three candidate biomarkers were found to be significantly different between the two groups, including lysophospholipids and sphingolipids related to inflammation; bile acids, tryptophan metabolites, and thyroxine, related to the metabolism of the gut microflora. Confirmation results demonstrated the specificity of major alterations occurring in ARDS patients with influenza A (H1N1) virus infection., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Humulus lupulus L., commonly named hop, is well-known for its sedative and estrogenic activity. While hop cones are widely characterized, only few works have been carried out on the young shoots of this plant. In the light of this, the aim of this study was to identify for the first time the flavonoids present in young hop shoots and to compare the composition of samples harvested from different locations in Northern Italy with their antioxidant activity. The samples were extracted by means of dynamic maceration with methanol. The HPLC-UV/DAD, HPLC-ESI-MS and MS 2 analysis were carried out by using an Ascentis C 18 column (250×4.6mm I.D., 5μm), with a mobile phase composed of 0.1M formic acid in both water and acetonitrile, under gradient elution. Quercetin and kaempferol glycosides were the main compounds identified and quantified in hop shoot extracts. Total flavonols ranged from 2698±185 to 517±48μg/g (fresh weight). The antioxidant activity was determined by means of the radical scavenging activity assay against diphenylpicrylhydrazyl (DPPH) and by using a photochemiluscence assay with a Photochem ® apparatus. The results showed that hop shoots represent a new source of flavonols; therefore, they can be useful for a possible incorporation in the diet as a functional food or applied in the nutraceutical ambit., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The present study was aimed at the development of a new analytical method for the comprehensive multi-component analysis of polyphenols in Punica granatum L. (pomegranate) juice and peel. While pomegranate juice was directly analysed after simple centrifugation, different extraction techniques, including maceration, heat reflux extraction, ultrasound-assisted extraction and microwave-assisted extraction, were compared in order to obtain a high yield of the target analytes from pomegranate peel. Dynamic maceration with a mixture of water and ethanol 80:20 (v/v) with 0.1% of hydrochloric acid as the extraction solvent provided the best result in terms of recovery of pomegranate secondary metabolites. The quali- and quantitative analysis of pomegranate polyphenols was performed by high-performance liquid chromatography with diode array and electrospray ionization-mass spectrometry detection. The application of fused-core column technology allowed us to obtain an improvement of the chromatographic performance in comparison with that of conventional particulate stationary phases, thus enabling a good separation of all constituents in a shorter time and with low solvent usage. The analytical method was completely validated to show compliance with the International Conference on Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human Use guidelines and successfully applied to the characterisation of commercial and experimental pomegranate samples, thus demonstrating its efficiency as a tool for the fingerprinting of this plant material. The quantitative data collected were submitted to principal component analysis, in order to highlight the possible presence of pomegranate samples with high content of secondary metabolites. From the statistical analysis, four experimental samples showed a notable content of bioactive compounds in the peels, while commercial ones still represent the best source of healthy juice., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Flavonoids represent a potential source of new antitrypanosomatidic leads. Starting from a library of natural products, we combined target-based screening on pteridine reductase 1 with phenotypic screening on Trypanosoma brucei for hit identification. Flavonols were identified as hits, and a library of 16 derivatives was synthesized. Twelve compounds showed EC50 values against T. brucei below 10 μM. Four X-ray crystal structures and docking studies explained the observed structure-activity relationships. Compound 2 (3,6-dihydroxy-2-(3-hydroxyphenyl)-4H-chromen-4-one) was selected for pharmacokinetic studies. Encapsulation of compound 2 in PLGA nanoparticles or cyclodextrins resulted in lower in vitro toxicity when compared to the free compound. Combination studies with methotrexate revealed that compound 13 (3-hydroxy-6-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one) has the highest synergistic effect at concentration of 1.3 μM, 11.7-fold dose reduction index and no toxicity toward host cells. Our results provide the basis for further chemical modifications aimed at identifying novel antitrypanosomatidic agents showing higher potency toward PTR1 and increased metabolic stability.

Essential oils (EOs) are known and used for their biological, antibacterial, antifungal and antioxidant properties. Numerous studies have shown that EOs exhibit a large spectrum of biological activities in vitro. The incidence of drug-resistant pathogens and the toxicity of antibiotics have drawn attention to the antimicrobial activity of natural products, encouraging the development of alternative treatments. The aim of this study was to analyse the phytochemical and the cytotoxic characteristic of 36 EOs; we then evaluated the antimicrobial activity of the less-toxic EOs on Gram-positive, Gram-negative and fungi strains. The results showed low cytotoxicity in seven EOs and good activity against Gram-negative and Candida spp. strains. Based on our results, EOs could be proposed as a novel group of therapeutic agents. Further experiments are necessary to confirm their pharmacological effectiveness, and to determine potential toxic effects and the mechanism of their activity in in vivo models.

Although general anesthetics are thought to modify critical neuronal functions, their impact on neuronal communication has been poorly examined. We have investigated the effect induced by desflurane, a clinically used general anesthetic, on information transfer at the synapse between mossy fibers and granule cells of cerebellum, where this analysis can be carried out extensively. Mutual information values were assessed by measuring the variability of postsynaptic output in relationship to the variability of a given set of presynaptic inputs. Desflurane synchronized granule cell firing and reduced mutual information in response to physiologically relevant mossy fibers patterns. The decrease in spike variability was due to an increased postsynaptic membrane excitability, which made granule cells more prone to elicit action potentials, and to a strengthened synaptic inhibition, which markedly hampered membrane depolarization. These concomitant actions on granule cells firing indicate that desflurane re-shapes the transfer of information between neurons by providing a less informative neurotransmission rather than completely silencing neuronal activity.

Gestational Diabetes Mellitus (GDM) is defined as glucose intolerance with onset or first recognition during pregnancy. It is affecting approximately up to 14% of all pregnancies with an increasing tendency. GDM has been related to relevant short-term and long-term health complications for both mother and offspring. Recent studies strongly emphasized the role of several essential amino acids in the pathogenesis of obesity and highlighted their strong correlation with insulin resistance, but there are no references related to modifications in D-AAs in biological fluids. As D-AA elimination proceeds mainly by renal excretion, urine was the selected sample to evaluate the alterations in free D-AAs ratio in a GDM study. Only 1 mL of first void urine or standard solution was required for purification, by using a Discovery DSC-SCX SPE cartridge (500 mg/3 mL) and derivatization into their N(O)-pentafluoropropionyl amino acid 2-propyl esters. Enantiomeric separation was carried out by GC-MS on a Chirasil-L-Val N-propionyl-L-valine-tert-butylamide polysiloxane fused-silica capillary column (25 m×0.25 mm I.D., 0.12 μm film thickness, Agilent Technologies, Waldbronn, Germany), under programmed temperature elution. Detection was performed with an ion trap mass analyzer, operating in the full scan mode in the m/z 50-350 range. 14 pairs of derivatives of D-and L-AAs were separated. The steps of sample preparation, derivatization and GC-MS conditions were optimized for both urine and standards. Several conditions affecting the SPE procedure, such as sorbent mass/volume ratio of the cartridge, sample dilution and pH, were optimized. Volume of reagents and solvents and reaction temperature and time were also tested for the derivatization. Regarding the GC-MS parameters, split ratio, temperature program and mass range were optimized. The final method was validated in terms of linearity, sensitivity, accuracy and precision for D-Ala, D-Pro, D-Ser, D-Met, D-Phe, D-Glu, D-Orn and D-Lys. Identification of AAs in urine samples was based on retention time and mass spectra. Urine from 20 women with GDM and 20 pregnant women with normal glucose tolerance (after 2-h 75-g oral glucose tolerance test), matched according to the week of gestation and age (22-28 week of gestation and age 24-37 years), were enrolled into the study. %D-Relative amounts were determined for Ala, Val, Thr, Ser, Leu, Asx (Asp+Asn), Glx (Glu+Gln), Met, Phe, Tyr, Orn and Lys. Statistically significant differences (p<0.05) were observed only for D-Phe and higher values were found in the GDM group. It is possible that D-Phe could be involved in metabolic/signaling pathways to compensate early stages of insulin resistance, although further work is necessary to confirm this hypothesis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Paronychia argentea Lam., belonging to the Caryophyllaceae family, is a perennial plant widely distributed in Algeria. Even though this plant is used in the Algerian popular medicine, its phytochemical characterization is incomplete. In this study, the flavonoid profile and the in vitro antioxidant activity of the ethanolic extract, decoction and infusion of P. argentea aerial parts are reported. Flavonoids were analyzed by means of high-performance liquid chromatography coupled with diode array detection and electrospray ionization mass spectrometry. Eleven compounds were identified and six of them, including isorhamnetin-3-O-dihexoside, quercetin-3-O-glucoside, quercetinmethylether-O-hexoside, quercetin, jaceosidin and isorhamnetin, were described in this plant for the first time. The ethanol extract showed the highest flavonoid content, followed by the decoction and the infusion (25.4 ± 0.8 mg/g of DM, 8.4 ± 0.5 mg/g of DM, 0.2 mg/g of DM, respectively), while the best antioxidant activity was shown by the decoction (RC0.5 = 178 μg/mL for reducing power, 72.4% of inhibition of lipid peroxidation, IC50 = 27.38μ g/mL for DPPH radical scavenging activity and 59.7% of inhibition of NO radical). These results showed that P. argentea decoction could be considered as a valuable source of flavonoids and antioxidants that might contribute to the valorization of the phytotherapeutic potential of this plant., (Copyright © 2015 Elsevier B.V. All rights reserved.)