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Tyrosinase is a target enzyme to be inhibited in order to reduce excessive melanin production and prevent typical age-related skin disorders. Essential oils are complex mixtures of volatile compounds, belonging mainly to monoterpenoids and sesquiterpenoids, which have been relatively little studied as tyrosinase inhibitors. Among the monoterpenoids, citral (a mixture of neral and geranial) is a fragrance compound in several essential oils that has shown interesting tyrosinase inhibitory activity. Although citral is listed as an allergen among the 26 fragrances in Annex III of the Cosmetics Directive 2003/15/EC, it can be safely used for the formulation of topical products in amounts that are not expected to cause skin sensitization, as shown by various commercially available products.The aim of this work was to evaluate two different formulations (oil/water emulsion, oily solution) containing a new combination of essential oils ( Litsea cubeba, Pinus mugo, Cymbopogon winterianus ) applied to the skin both in nonocclusive and partially occlusive modes. The blend is designed to reduce the concentration of citral to avoid potential skin reactions while taking advantage of the inhibitory activity of citral. Specifically, the amount of citral and other bioactive compounds (myrcene, citronellal) delivered through the skin was studied as a function of formulation and mode of application.The results show that an oil/water emulsion is preferable because it releases the bioactive compounds rapidly and minimizes their evaporative loss. In addition, semi-occluded conditions are required to prevent evaporation, resulting in higher availability of the bioactive compounds in viable skin., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

The wide range of applications of hemp products, together with the environmental benefits that come from hemp cultivation are driving up the market demand for Cannabis sativa L. plant. One of the main restrictions for hemp cultivation and marketing concerns the content of delta-9-tetrahydrocannabidiol (Δ 9 -THC), which is known to have psychotomimetic effect. If the recent growing of hemp market is beneficial by an economic and environmental point of view, it is necessary to develop reliable analytical methods for the chemical characterization of hemp products, to guarantee the safety of use for the customers. This study aimed to develop a simple ultrasound-assisted dispersive solid-liquid microextraction (UA-DSLME) method for the extraction of cannabinoids in hemp products, using eutectic solvents (ESs) as extraction material. Two types of ESs were compared: one prepared with a [Ch + ][Br - ]-modified salts as hydrogen bond acceptor and one based on natural terpenoids. The ultrasound-assisted dispersive solid-liquid microextraction method was optimized to be applied for the analysis of aerial parts of hemp collected before flowering, hemp inflorescences and a commercial sample called CBD oil, and proved to be robust and versatile. Under optimal conditions, only 100 µL of ES and 2 mL of water as co-solvent were used in the US-assisted extraction, before the analysis in the UHPLC-PDA system. The developed approach allowed to obtain the same chemical profile of conventional methods, while improving the greenness of the method and the enrichment of the marker analytes. To overcome the strong matrix effect for cannabinoids, a matrix-matched calibration was used. Blank matrices of the samples under study were easily obtained by performing an exhaustive extraction of the marker analytes in the hemp samples. These matrices were successfully used for validation, achieving accuracy values between 82% and 118%., 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 © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Type 2 diabetes (T2D) is a progressive metabolic disorder of glucose metabolism. One of the therapeutic approaches for the treatment of T2D is reducing postprandial hyperglycaemia through inhibition of the digestive enzymes α-glucosidase and α-amylase. In this context, aimed at identifying natural products endowed with anti-T2D potential, we focused on Ptilostemon casabonae (L.) Greuter, a species belonging to Asteraceae family. Enzymatic inhibition, antioxidant activity, phenolic composition and cellular assays were performed. This study revealed that the P. casabonae hydroalcoholic extract exerts a potent inhibitory activity against α-glucosidase. This activity is supported by an antioxidant effect, preventing ROS formation in a stressed cellular system. HPLC-PDA-MS/MS analysis, revealed a complex polyphenolic fraction. Among the tested pure compounds, 1,5-dicaffeoylquinic acid, apigenin and rutin displayed good α-glucosidase inhibitory activity. Our study suggested new potential of P. casabonae encouraging us to further testing the possible therapeutic potential of this extract.

Elettaria cardamomum (L.) Maton (Zingiberaceae family) is a plant traditionally used in Ayurvedic and Chinese medicine. In this work, the essential oil of E. cardamomum was found to inhibit the enzymes AChE (62.6% of inhibition, IC 50 24.9 μg/mL) and BChE (55.8% of inhibition, IC 50 25.9 μg/mL) by performing an in vitro colorimetric assay using the Ellman method. A bio-guided fractionation approach was used to isolate fractions/pure compounds that were tested individually to evaluate their activity. The resulting oxygenated fraction was found to be active against both AChE (percentage inhibition 42.8%) and BChE (percentage inhibition 63.7%), while the hydrocarbon fraction was inactive. The activity was attributed to a pool of oxygenated terpenes (α-terpinyl acetate, 1,8-cineole, linalool, linalyl acetate, and α-terpineol) that synergistically contributed to the overall activity of the essential oil.

Quality control of essential oils is fundamental for verifying their authenticity and conformity with quality standards, ensuring their safety and regulatory compliance, and monitoring their consistency. Companies that produce or market essential oils routinely evaluate the quality and authenticity of their products. However, they also must deal with increasing attention to environmental sustainability as well as practical considerations such as productivity, cost, and simplicity of methods. In this study, enantioselective gas chromatography (GC) was adopted to evaluate the quality of sweet and bitter orange essential oils, used as a case study. The analytical conditions were optimized and translated to fast GC to evaluate the impact of this approach on the environmental footprint of the analyses. The greenness of fast GC, compared with conventional GC, was quantitatively evaluated using a dedicated metric tool (AGREE), and important improvements have been calculated. The developed methods were applied to a set of commercial essential oils, and the data about the enantiomeric composition and relative percentage abundance were elaborated through multivariate statistics (principal component analysis). The results showed that fast chiral gas chromatography enables the classification of citrus essential oil samples and can be considered an environmentally friendly and sustainable approach for evaluating their quality.

The cardueae are a common species in the Mediterranean area where they grow spontaneously and are traditionally employed as food and for health purposes. In this work, five Cardueae, including two sub-endemic species (four Carduus and three Ptilostemon casabonae (L.) Greuter samples from different locations) were collected from Sardinia and the Corse islands. All the considered plants are characteristic of the area, in particular the sub-endemic species C. cephalanthus and P. casabonae . This work aims to obtain, for the first time, the amino compounds profile (primary metabolites) of these little-studied species to detect for any similarities and differences among the different samples using statistical analyses. A recently developed method was employed, where diethyl ethoxymethylenemalonate (DEEMM) derivatives are detected in a neutral loss scan mode using high performance liquid chromatography in tandem with a mass spectrometry technique. In total, 42 amino compounds were detected, of which 33 were fully identified and semi-quantified. Overall, the results show that DEEMM-derivatized amino compounds are qualitatively similar among the considered samples. Nonetheless, a discrimination at the genus level is possible. This work adds more information regarding the phytochemical composition regarding the primary metabolites of the considered samples, their discriminations and the search for compounds with potential health benefits.

Acrylamide (AA) is a product of food heating process that is widely present in cooked foods and known to be toxic to humans. Exposure data has revealed coffee to be one of the sources of this toxicant in adult diets. A great deal of effort has been invested into finding ways of reducing AA formation during coffee processing. However, despite the accumulated knowledge and mitigation strategies applied so far, AA reduction in coffee is still a challenge compared to other heat-processed foods in which the wider raw-material selection and progress in technological processes and/or changes in the recipes are possible at the industrial level. This review presents a critical analysis of the accumulated knowledge on the formation of AA in coffee as well as on the mitigation strategies that have been investigated to date, with a focus on current applicability in industry and little explored topics., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Phosphonium-based ionic liquids (ILs) have proven to be successful stationary phases (SPs) for gas chromatography (GC) in several fields of application because of their unique selectivity and good chromatographic properties. This study focuses on the use of two ILs as GC SPs that are based on the phosphonium derivatives trihexyl(tetradecyl)phosphonium chloride ([P 66614 + ] [Cl - ]), and trihexyl(tetradecyl)phosphonium bis[(trifluoromethyl)sulfonyl]imide ([P 66614 + ][NTf 2 - ]), which have previously been shown to be complementary in terms of chromatographic selectivity and retention. Their application in routine analysis has been limited by their lower maximum allowable operating temperatures (MAOT) (200 °C for the [P 66614 + ][Cl - ] IL and 180 °C for [P 66614 + ][NTf 2 - ]), which restricts their use to samples that consist of analytes with relatively high volatility. A previous study carried out in the Authors' laboratory focused on extending the use of the [P 66614 + ][Cl - ] IL SP to the analysis of samples with analytes of medium-to-low volatility by optimizing column characteristics and operative conditions. This study addresses the immobilization of both the [P 66614 + ][Cl - ] and [P 66614 + ][NTf 2 - ] ILs to the inner wall of fused silica columns to increase their MAOT under soft and hard reaction conditions. The resulting MAOT depended on more or less drastic immobilization conditions, and reached 220 °C for soft immobilization (So-Im) and 240 °C for hard immobilization (Ha-Im) in the [P 66614 + ][Cl - ] IL columns, and 200 °C for So-Im and 220° for Ha-Im in columns coated with the [P 66614 + ] [NTf 2 - ] IL. The influence of immobilization on the separation power and performance of all the columns has been evaluated using i) the Grob test, ii) a model mixture of 41 compounds of different polarity, structure, and with different organic functional groups representative of the flavor and fragrance field, iii) a standard mixture of 37 fatty acid methyl esters, iv) the peppermint essential oil, v) two mixtures of sesquiterpenic alcohols (farnesols and santalols), and vi) a standard mixture of 16 pesticides. These test samples were also used to demonstrate the complementarity of the two phosphonium-based IL SPs in terms of selectivity and retention., 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. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Currently, there is a renewed interest in cannabis-related products in different fields because of the rich phytocomplex of this plant, together with its fiber and agricultural features. In this context, the current study aims to chemically characterize different samples of fiber-type Cannabis sativa L. grown in Italy as a potential health promoting source. An ultrasound-assisted solid-liquid extraction (UA-SLE) method was first developed and optimized to obtain a fingerprinting of the investigated phytocomplex. Analyses were carried out through an ultra high performance liquid chromatography equipped with a photodiode array detector in series with triple quadrupole system with an electrospray ionization (ESI) interface (UHPLC-UV-ESI-MS/MS) and showed that the phytocomplex mainly includes flavonoids and non-psychotomimetic cannabinoids. The method was then applied to characterize and compare 24 samples of fiber-type Cannabis sativa L. aerial parts (mainly stems and leaves), which differed for the growth stages (from mid-vegetative to early flowering), growth land plots, and methods of drying (forced-draft oven or freeze-drying). The quali-quantitative analysis showed that a freeze-drying method seems to better preserve the chemical composition of the samples, while the location of the land plot and the growth stage of the plant (which did not comprise inflorescences) had minor influences on the chemical pattern. These results were also supported by spectrophotometric in-vitro assays (scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH • ) and 2,2'-azinobis-3-ethyl-benzthiazoline-6-sulphonate (ABTS +• ) radicals and inhibitory activity against tyrosinase and elastase enzymes) to investigate the potential biological activity of these samples and the contribution of non-psychotomimetic cannabinoids.

This review is an overview of the recent advances of gas chromatography in essential oil analysis; in particular, it focuses on both the new stationary phases and the advanced analytical methods and instrumentations. A paragraph is dedicated to ionic liquids as gas chromatography stationary phases, showing that, thanks to their peculiar selectivity, they can offer a complementary contribution to conventional stationary phases for the analysis of complex essential oils and the separation of critical pairs of components. Strategies to speed-up the analysis time, thus answering to the ever increasing request for routine essential oils quality control, are also discussed. Last but not least, a paragraph is dedicated to recent developments in column miniaturization in particular that based on microelectromechanical-system technology in a perspective of developing micro-gas chromatographic systems to optimize the energy consumption as well as the instrumentation dimensions. A number of applications in the essential oil field is also included., (© 2021 Wiley-VCH GmbH.)

Amino compounds, such as amino acids and biogenic amines, are important metabolites that can be found in diverse natural matrices. The most common method for amino compound analysis nowadays is reversed-phase liquid chromatography tandem mass spectrometry (RPLC-MS/MS). However, due to the polar and the basic nature of amines, their RPLC retention is often insufficient or peaks are tailing. Derivatization is a way to overcome the issue and in the present work amino compounds are derivatized with diethyl ethoxymethylenemalonate (DEEMM) and analyzed by a RPLC triple quadrupole MS system in neutral loss scan (NLS) mode (loss of 46). This allows to target all compounds in the sample that undergo derivatization with DEEMM, so that the amino compound profile of the sample is obtained. To the best of our knowledge, the NLS acquisition mode has never been employed to target amino compounds after DEEMM derivatization. In the first part of the study, eight amino acids (arginine, aspartic acid, threonine, proline, tyrosine, tryptophan, phenylalanine and isoleucine) were employed as model compounds for method optimization, with good results in terms of DEEMM derivatives detection and repeatability. The developed method was successfully applied to a complex extract from the plant species Carduus nutans subsp. macrocephalus (Desf.) Nyman, with 18 amino acids and 3 other amines being identified. The proposed approach could be employed for straightforward identification of known and unknown amino compounds in different types of matrices., 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. Published by Elsevier B.V.)

In a search for natural compounds with anti-HIV-1 activity, we studied the effect of the ethanolic extract obtained from leaves, bark, and peels of Punica granatum L. for the inhibition of the HIV-1 reverse transcriptase (RT)-associated ribonuclease H (RNase H) and integrase (IN) LEDGF-dependent activities. The chemical analyses led to the detection of compounds belonging mainly to the phenolic and flavonoid chemical classes. Ellagic acid, flavones, and triterpenoid molecules were identified in leaves. The bark and peels were characterized by the presence of hydrolyzable tannins, such as punicalins and punicalagins, together with ellagic acid. Among the isolated compounds, the hydrolyzable tannins and ellagic acid showed a very high inhibition (IC 50 values ranging from 0.12 to 1.4 µM and 0.065 to 0.09 µM of the RNase H and IN activities, respectively). Of the flavonoids, luteolin and apigenin were found to be able to inhibit RNase H and IN functions (IC 50 values in the 3.7-22 μM range), whereas luteolin 7- O -glucoside showed selective activity for HIV-1 IN. In contrast, betulinic acid, ursolic acid, and oleanolic acid were selective for the HIV-1 RNase H activity. Our results strongly support the potential of non-edible P. granatum organs as a valuable source of anti-HIV-1 compounds.

The quality control of essential oils (EO) principally aims at revealing the presence of adulterations and at quantifying compounds that are limited by law by evaluating EO chemical compositions, usually in terms of the normalised relative abundance of selected markers, for comparison to reference values reported in pharmacopoeias and/or international norms. Common adulterations of EO consist of the addition of cheaper EO or synthetic materials. This adulteration can be detected by calculating the percent normalised areas of selected markers or the enantiomeric composition of chiral components. The dilution of the EO with vegetable oils is another type of adulteration. This adulteration is quite devious, as it modifies neither the qualitative composition of the resulting EO nor the marker's normalised percentage abundance, which is no longer diagnostic, and an absolute quantitative analysis is required. This study aims at verifying the application of the two above approaches (i.e., normalised relative abundance and absolute quantitation) to detect EO adulterations, with examples involving selected commercial EO (lavender, bergamot and tea tree) adulterated with synthetic components, EO of different origin and lower economical values and heavy vegetable oils. The results show that absolute quantitation is necessary to highlight adulteration with heavy vegetable oils, providing that a reference quantitative profile is available.

Accurate, reliable, and informative mapping of untargeted and targeted components across many samples is here performed by combining off-line GC-Olfactometry (GC-O) and comprehensive two-dimensional gas chromatography (GC×GC) coupled to time-of-flight mass spectrometry with variable ionization energy (TOF MS featuring Tandem Ionization™). In particular, untargeted and targeted (UT) features patterns are processed by chromatographic fingerprinting, giving differential priority to potent odorants' retention-times regions. Distinguishing peppermint essential oil (EO) from Piedmont (Italy - Mentha × piperita L. var. Italo-Mitcham - Menta di Pancalieri EO), with its unique sensory fingerprint (i.e., freshness and long-lasting sweetness), from high-quality peppermint EOs produced in other areas poses a great challenge. Chromatographic UT fingerprinting provided a great chemical dimensionality by mapping more than 350 peak-regions at 70 eV and 135 at 12 eV. From them, 95 components were identified and responses compared to available literature. Then, potent odorants, detected by GC-O using the aroma extraction dilution analysis (AEDA), were tracked over the chromatographic space and tentatively identified. With the highest flavor dilution (FD), 1,8-cineole (eucalyptus, fresh, camphoraceous); menthone (minty, herbaceous); and menthofuran (minty, musty, petroleum-like) were highlighted. Responsible for creamy and coumarinic notes were the diasteroisomers of (3,6)-dimethyl-4,5,6,7-tetrahydrobenzo[b]-furan-2(3H)-one (i.e., menthofurolactones), detected in higher relative abundance in Pancalieri EOs. By prioritizing the investigation of volatiles on higher LogFD retention regions, including 131 untargeted/targeted features, Pancalieri EOs were separately clustered from United States samples. Besides pre-targeted analytes, additional untargeted features were post-processed for identification within marker chemicals. Myrtenyl methyl ether, ethyl 3-methyl butanoate, propyl-2-methylbutanoate, and (E)-2-hexenal were putatively identified. Of the "unknown - knowns" with diagnostic roles, all metadata were collected including low energy spectra at 12 eV, which were found to be highly complementary to 70 eV spectra., 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. Published by Elsevier B.V.)

Excessive melanin production causes serious dermatological conditions as well as minor aesthetic problems (i.e., freckles and solar lentigo). The downregulation of tyrosinase is a widespread approach for the treatment of such disorders, and plant extracts have often proven to be valuable sources of tyrosinase inhibitors. Citral (a mixture of neral and geranial) is an important fragrance ingredient that has shown anti-tyrosinase potential. It is highly concentrated in the essential oils (EOs) of Cymbopogon schoenanthus (L.) Spreng., Litsea cubeba (Lour.) Pers., Melissa officinalis L., and Verbena officinalis L. However, only L. cubeba EO has been investigated for use as a potential skin-whitening agent. This work evaluates the in vitro tyrosinase inhibitory activity of these EOs and studies, using bio-assay oriented fractionation, whether their differing chemical compositions influence the overall EO inhibitory activities via possible synergistic, additive, and/or competitive interactions between EOs components. The inhibitory activity of C. schoenanthus EO and that of M. officinalis EOs, with negligible (+)-citronellal amounts, were in-line with their citral content. On the other hand, L. cubeba and V. officinalis EOs inhibited tyrosinase to considerably greater extents as they contained β-myrcene, which contributed to the overall EO activities. Similar observations were made for M. officinalis EO, which bears high (+)-citronellal content which increased citral activity.

Biogenic volatile organic compounds (BVOCs) are metabolites emitted by living plants that have a fundamental ecological role since they influence atmospheric chemistry, plant communication and pollinator/herbivore behaviour, and human activities. Over the years, several strategies have been developed to isolate and identify them, and to take advantage of their activity. The main techniques used for in-vivo analyses include dynamic headspace (D-HS), static headspace (S-HS) and, more recently, direct contact (DC) methods in association with gas chromatography (GC) and mass spectrometry (MS). The aim of this review is to provide insight into the in-vivo characterisation of plant volatile emissions with a focus on sampling, analysis and possible applications. This review first provides a critical discussion of the challenges associated with conventional approaches and their limitations and advantages. Then, it describes a series of applications of in-vivo volatilomic studies to enhance how the information they provide impact on our knowledge of plant behaviour, including the effects of abiotic (damage, flooding, climate) and biotic (insect feeding) stress factors in relation to the plants., 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.)

Zika virus, an arthropod-borne flavivirus, is an emerging healthcare threat worldwide. Zika virus is responsible for severe neurological effects, such as paralytic Guillain-Barrè syndrome, in adults, and also congenital malformations, especially microcephaly. No specific antiviral drugs and vaccines are currently available, and treatments are palliative, but medicinal plants show great potential as natural sources of anti-Zika phytochemicals. This study deals with the investigation of the composition, cytotoxicity, and anti-Zika activity of Punica granatum leaf ethanolic extract, fractions, and phytoconstituents. P. granatum leaves were collected from different areas in Italy and Greece in different seasons. Crude extracts were analyzed and fractionated, and the pure compounds were isolated. The phytochemical and biomolecular fingerprint of the pomegranate leaves was determined. The antiviral activities of the leaf extract, fractions, and compounds were investigated against the MR766 and HPF2013 Zika virus strains in vitro . Both the extract and its fractions were found to be active against Zika virus infection. Of the compounds isolated, ellagic acid showed particular anti-Zika activities, with EC 50 values of 30.86 µM for MR766 and 46.23 µM for HPF2013. The mechanism of action was investigated using specific antiviral assays, and it was demonstrated that ellagic acid was primarily active as it prevented Zika virus infection and was able to significantly reduce Zika virus progeny production. Our data demonstrate the anti-Zika activity of pomegranate leaf extract and ellagic acid for the first time. These findings identify ellagic acid as a possible anti-Zika candidate compound that can be used for preventive and therapeutic interventions., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by unpaired blood glycaemia maintenance. T2DM can be treated by inhibiting carbohydrate hydrolyzing enzymes (α-amylases and α-glucosidases) to decrease postprandial hyperglycemia. Acarbose and voglibose are inhibitors used in clinical practice. However, these drugs are associated with unpleasant gastrointestinal side effects. This study explores new α-amylase inhibitors deriving from plant volatile specialized metabolites. Sixty-two essential oils (EOs) from different plant species and botanical families were subjected to α-amylase in vitro enzymatic assay and chemically characterized using gas chromatography coupled to mass spectrometry. Several EOs were found to be potential α-amylase inhibitors, and Eucalyptus radiata , Laurus nobilis , and Myristica fragrans EOs displayed inhibitory capacities comparable to that of the positive control (i.e., acarbose). A bio-guided fractionation approach was adopted to isolate and identify the active fractions/compounds of Eucalyptus radiata and Myristica fragrans EOs. The bio-guided fractionation revealed that EOs α-amylase inhibitory activity is often the result of antagonist, additive, or synergistic interactions among their bioactive constituents and led to the identification of 1,8-cineole, 4-terpineol, α-terpineol, α-pinene, and β-pinene as bioactive compounds, also confirmed when they were tested singularly. These results demonstrate that EO oils are a promising source of potential α-amylase inhibitors.

The Echinacea genus includes a number of species that are commercially employed for the preparation of herbal products. Echinacea angustifolia DC. is one of these and is widely used, mainly for its immunomodulating properties, as it contains a wide range of compounds that belong to different chemical classes. In particular, echinacoside, cynarin and lipophylic alkylamides are the main specialized metabolites of the roots and can be considered to be marker compounds. In this work, 65 E. angustifolia accessions have been compared in a field trial in Italy, with the aim of investigating the variability/stability of the weight and chemical composition of their roots in order to identify the accessions that are most promising for future genetic-improvement programs. The morphological characteristics of the aerial parts have also been investigated. Seventeen samples were discarded due to germination or plantlet-development issues. Seven of the remaining accessions were identified as being different Echinacea species after a combined phytochemical and morphological evaluation. The morphological traits of the epigeal part, the root weight and the chemical composition data of the 41 confirmed E. angustifolia accessions were submitted to multivariate statistical analysis and a moderately homogenous sample distribution, with low selected-marker variability, was observed. Good echinacoside content was detected in almost all roots (>0.5%). However, two groups of accessions stood out because of their interesting features: One group possessed small roots, but had a high concentration of marker compounds, while another had highly developed roots and a good amount of marker compounds. These accessions can therefore be exploited for future selection work.

This paper proposes a new sustainable and simple strategy for the micro-scale extraction of phenolic compounds from grapevine leaves with analytical purpose. The method is based on a microwave-assisted solid-liquid extraction approach (MA-SLE), using an aqueous solution of an ionic liquid (IL)-based surfactant as extraction phase. The method does not require organic solvents, nor any clean-up step, apart from filtration prior to the injection in the analytical system. Two IL-based surfactants were evaluated, and the method was optimized by using experimental designs, resulting in the use of small amounts of sample (100 mg) and extraction phase (2.25 mL), low concentrations of the selected 1-hexadecyl-3-butyl imidazolium bromide IL (0.1 mM), and 30 min of extraction time. The proposed methodology was applied for the determination of the polyphenolic pattern of six different varieties of Vitis vinifera leaves from the Canary Islands, using high-performance liquid chromatography and photodiode array detection for the quantification of the compounds. The proposed MA-SLE approach was greener, simpler, and more effective than other methods, while the results from the analysis of the leaves samples demonstrate that these by-products can be exploited as a source of natural compounds for many applications.

Room temperature ionic liquids (ILs) are well established stationary phases (SPs) for gas chromatography (GC) in several fields of applications because of their unique and tunable selectivity, low vapor pressure and volatility, high thermal stability (over 300 °C), and good chromatographic properties. This study is focused on an IL based on a phosphonium derivative (trihexyl(tetradecyl)phosphonium chloride, [P 66614 + ] [Cl - ]), previously shown to be suitable as a gas chromatographic SP because of its unique selectivity. In particular, it aims to establish the operative conditions to apply [P 66614 + ][Cl - ] to routine analysis of samples containing medium to high volatility analytes with different polarity, organic functional groups and chemical structure. In the first part, the study critically evaluates long term [P 66614 + ][Cl - ] column stability and maximum allowable operating temperatures (MAOT). The relatively low MAOT (210 °C) requires the adoption of a dedicated approach for analytes eluting above this temperature based on a suitable combination of efficiency and selectivity, and column characteristics (length, inner diameter and film thickness) and operative conditions. The performance of [P 66614 + ][Cl - ] as a GC SP have been validated through the Grob test, a model mixture of 41 compounds of different polarity, structure, and with different organic functional groups in the flavor and fragrance field, a standard mixture of 37 fatty acid methyl esters, some essential oils containing pairs or groups of compounds of different volatility critical to separate in particular peppermint, thyme, oregano, sandalwood and frankincense. The above approach has produced highly satisfactory separations with all of the samples investigated., 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. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests, (Copyright © 2020 Elsevier B.V. All rights reserved.)

Here we focus on the highly conserved MYB-bHLH-WD repeat (MBW) transcriptional complex model in eggplant, which is pivotal in the transcriptional regulation of the anthocyanin biosynthetic pathway. Through a genome-wide approach performed on the recently released Eggplant Genome (cv. 67/3) previously identified, and reconfirmed by us, members belonging to the MBW complex (SmelANT1, SmelAN2, SmelJAF13, SmelAN1) were functionally characterized. Furthermore, a regulatory R3 MYB type repressor (SmelMYBL1), never reported before, was identified and characterized as well. Through a qPCR approach, we revealed specific transcriptional patterns of candidate genes in different plant tissue/organs at two stages of fruit development. Two strategies were adopted for investigating the interactions of bHLH partners (SmelAN1, SmelJAF13) with MYB counterparts (SmelANT1, SmelAN2 and SmelMYBL1): Yeast Two Hybrid (Y2H) and Bimolecular Fluorescent Complementation (BiFC) in A. thaliana mesophylls protoplast. Agro-infiltration experiments highlighted that N. benthamiana leaves transiently expressing SmelANT1 and SmelAN2 showed an anthocyanin-pigmented phenotype, while their co-expression with SmelMYBL1 prevented anthocyanin accumulation. Our results suggest that SmelMYBL1 may inhibits the MBW complex via the competition with MYB activators for bHLH binding site, although this hypothesis requires further elucidation., Competing Interests: The authors have declared that no competing interests exist.

The frankincense resins, secreted from Boswellia species, are an uncommon example of a natural raw material where every class of terpenoids is present in similar proportions. Diterpenoids (serratol, incensole, and incensole acetate) are used to discriminate samples from different species and origins. Headspace solid-phase microextraction has been used for frankincense analysis, although it requires long sampling time for medium- to low-volatility markers; headspace solid-phase microextraction under vacuum can overcome this limit. Gas chromatography is used for analysis but the separation of incensole and serratol needs polar stationary phases. In this study, we develop a method to discriminate frankincenses based on vacuum-assisted headspace solid-phase microextraction combined with fast gas chromatography-mass spectrometry with ionic liquid-based stationary phases. The optimized conditions for solid samples were: air evacuation below 0°C, 15 min of incubation time, and 15 min of extraction time. Losses of volatiles due to vial air-evacuation in the presence of the sample were minimized by sample amount above 100 mg and low sample temperature. Fast gas chromatography provides the baseline separation of all markers in 20 min. By applying vacuum sampling and fast gas chromatography, the total analysis was reduced to 50 min compared to 120 min (60 min sampling plus 60 min analysis) as previously reported. The method was successfully applied to commercial frankincense samples., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Melaleuca alternifolia essential oil (tea tree oil) is widely used as an ingredient in skin care products because of its recognized biological activities. The European Scientific Committee on Consumer Products constantly promotes research and collection of data on both skin distribution and systemic exposure to tea tree oil components after the application of topical formulations. This study quantitatively evaluates permeation, skin layer distribution ( stratum corneum , epidermis, and dermis), and release into the surrounding environment of bioactive tea tree oil markers (i.e., α -pinene, β -pinene, α -terpinene, 1,8-cineole, γ -terpinene, 4-terpineol, α -terpineol) when a 5% tea tree oil formulation is applied at a finite dosing regimen. Permeation kinetics were studied in vitro on pig ear skin using conventional static glass Franz diffusion cells and cells ad hoc modified to monitor the release of markers into the atmosphere. Formulation, receiving phases, and skin layers were analyzed using a fully automatic and solvent-free method based on headspace solid-phase microextraction/gas chromatography-mass spectrometry. This approach affords, for the first time, to quantify tea tree oil markers in the different skin layers while avoiding using solvents and overcoming the existing methods based on solvent extraction. The skin layers contained less than 1% of each tea tree oil marker in total. Only oxygenated terpenes significantly permeated across the skin, while hydrocarbons were only absorbed at trace level. Substantial amounts of markers were released into the atmosphere., Competing Interests: The authors declare that they have no conflict of interest., (Georg Thieme Verlag KG Stuttgart · New York.)

Cocoa smoky off-flavor is due to inappropriate post-harvest processing and cannot be removed in the subsequent chocolate-manufacturing steps. To date, no reliable analytical method to detect key-analytes responsible for smoky off-flavor in incoming raw material is available. This study aims to develop an analytical method, suitable for quality control, to detect smoky markers. The cocoa volatilome was first profiled by headspace solid phase microextration combined with comprehensive two-dimensional gas chromatography-mass spectrometry from a set of representative smoky and non-smoky samples; advanced fingerprinting revealed the chemicals responsible for the off-flavor. The results served to develop a 1D-GC method suitable for routine application. Ten identified smoky markers were subjected to accurate quantification, thereby defining operative ranges to accept/reject incoming bean samples. On average, these markers are present in smoky samples at 7 to 125 fold concentrations vs. those in non-smoky beans, ranging from 32.5 ng/g for naphtalene to 721.8 ng/g for phenol., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Infections caused by HSV-2 are a public health concern worldwide, and there is still a great demand for the discovery of novel anti-herpes virus agents effective against strains resistant to current antiviral agents. In this context, medicinal plants represent an alternative source of active compounds for developing efficient antiviral therapies. The aim of this study was to evaluate the antiviral activity of Arisaema tortuosum , a plant used in the traditional medicine of India. A chloroform soluble fraction of the leaves exhibited anti-HSV-2 activity with a selectivity index of 758. The extract was also active against acyclovir-resistant HSV-2 and HSV-1. The mechanism of action of the extract was investigated evidencing inhibition of both early and late events of the HSV-2 replicative cycle. A HPLC-PDA-MS/MS analysis showed the presence of flavonoids including apigenin and luteolin in the chloroform extract (CE). Apigenin and luteolin showed a high inhibitory activity with EC 50 values of 0.05 and 0.41 µg/mL, respectively. Both compounds exhibited antiviral activity when added up to 6 h post infection and were able to reduce the viral progeny production. In addition, apigenin interfered with cell-to-cell virus spread., Competing Interests: The authors declare that they have no conflict of interest., (Georg Thieme Verlag KG Stuttgart · New York.)

Green pruning residues (GPRs) and leaves from 16 red and white Vitis vinifera L. cultivars from Piedmont (Italy) were studied. The investigated samples were extracted by ultrasound-assisted extraction optimized by an experimental design, and quali- and quantitatively analyzed by HPLC-PDA-MS/MS. GPRs and leaves show a similar polyphenolic pattern, with quercetin 3- O -glucuronide, caftaric acid, and quercetin 3- O -glucoside as the main components, although in variable proportions. The HPLC results were related to the antioxidant activity, measured as total phenolic content and through DPPH and ABTS assays with similar results. Colorimetric in vitro assays, offline combined with HPLC-PDA analysis, determine which compounds contribute to the antioxidant activity in terms of radical scavenging abilities. Valorization of GPRs is a potential source of natural compounds that could be of interest in the health field, increasing their economic value together with a positive effect on the environment., Competing Interests: The authors declare no conflict of interest.

Qualitative and quantitative determination of volatile markers in aqueous based fragrances assumes ever-increasing importance, because of both the need for quality control and the safety-regulatory limitations introduced for several compounds. This study reports and critically discusses the results of applying new water-compatible ionic-liquid (IL) GC stationary phases, based on phosphonium and imidazolium derivative cations combined with trifluoromethanesulphonate (Watercol™) to the direct quantitative analysis of aqueous samples in the perfume field with GC-MS. Narrow-bore columns of different lengths, especially prepared for this study, were adopted to minimize the amount of water reaching the MS detector after GC separation. All GC-MS analysis steps were investigated, to achieve results compatible with quality control requirements for the volatiles of interest in this field, in terms of LODs, LOQs, and repeatability. Reliability of the GC-MS results was demonstrated by determining volatile allergens in two commercial perfumes, as per EU regulations concerning no-declaration limits for leave-on (0.001%) and rinse-off (0.01%) cosmetic products., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This study reports a follow-up investigation on the capture of specific DNA sequences using ion-tagged oligonucleotides (ITOs) and magnetic ionic liquids (MIL). Five allylimidazolium salts bearing octyl substituents ([AOIM + ]-ITOs) were used for the selective extraction of the internal transcribed spacer region (ITS) from Arabidopsis thaliana. In this work, the ability of the [AOIM + ]-ITOs to enhance the extraction of longer target sequences (~ 700 bp) of plant origin was shown. Moreover, the independence of the probe binding position and the importance of complementarity to the target region for the extraction performance were demonstrated. To test the specificity of the ITOs, the same experiments were performed using the ITS region from another plant species, with a lower target capture for the probes which were specific for the A. thaliana sequence. Finally, extraction in the presence of interferences (heterogenous DNA, primary and secondary metabolites, proteins) provided interesting and insightful results. This work illustrates the feasibility and versatility of these probes when coupled to MILs for rapid, cost-effective, and environmentally sensitive sample preparation in the extraction of specific target sequences from different origins. Graphical abstract.

A correct botanical identification and analytical quality control of volatile key-markers responsible for aroma and biological activities is necessary to monitor volatile compounds transferred from a plant to the related herbal tea and human intake to guarantee their safe use. This is mainly true for markers limited by regulations or by a recommended maximum amount of consumption per day. GC-MS is the elective technique to analyze volatiles, provided that for aqueous samples (herbal teas) an appropriate sample preparation procedure, and/or a water-compatible GC stationary phases are applied. Solid Phase Micro Extraction (SPME) on-line coupled to GC-MS in a fully automatic approach is here applied to sample and quantify key markers in plant material (headspace) and in the corresponding herbal tea (direct immersion). In parallel, a new generation of GC columns coated with ionic liquid based stationary phases compatible with aqueous samples (Watercol ™ ) was applied to test direct injection of aqueous samples (DAI-GC-FID). The latter approach fully bypasses sample preparation thus speeding up quality control. This study deals with the quantitation of menthol, α- and β-thujone, estragole, and anethole contained in several plant species commonly used for herbal teas (i.e. peppermint, sage, wormwood, fennel, aniseed) and regulated by International Organizations. The two methods gave comparable results and are characterized by high repeatability, linearity and accuracy, although, as expected, their sensitivity was different because DAI-GC-FID implies injection of the sample as such without analyte concentration as for DI-SPME-GC-MS. For instance, LOD and LOQ of estragole were 0.03 and 0.1 mg L -1 with DI-SPME-GC-MS and 0.1 and 0.8 mg L -1 with DAI-GC-FID. The two methods are fully complementary and their adoption depends on the amount of marker(s) to be quantified., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Ptilostemon casabonae (L.) Greuter is a Mediterranean endemism traditionally used for its health-giving properties. Little is known about this species, therefore this study provides additional information about the phytochemical and biomolecular patterns of this plant, to have a combined fingerprint as a taxonomic tool. Several P. casabonae specimens were therefore collected from three different sites, two from Sardinia (Italy) and one from Corsica and the hydroalcoholic extracts of their aerial parts were investigated through HPLC-PDA-MS/MS analysis to study the phenolic composition. Quercetin, luteolin, kaempferol, apigenin and diosmetin O-glycosides, and caffeoylquinic acid derivatives were found as main components. Samples from the three sites showed similar phenolic profiles, although statistical analyses highlighted some quantitative differences for several compounds. The biomolecular analysis included amplification and sequencing of ITS, 5S-rRNA-NTS and psbA regions. No difference was found in the nucleotides among the P. casabonae samples from different geographical origins; however, a comparison with other Ptilostemon species sequences from Genbank, revealed an interspecific variability of ITS and psbA regions. The combination of the results of the phytochemical and biomolecular studies provide information on P. casabonae useful to depict this little-known plant, which can also be applied for future investigations and to obtain a fingerprint of it. Moreover, the stability of the phenolic profile within the species affords to identify a set of specialised metabolites useful for its chemotaxonomic characterization. At the same time, the stability of the biomolecular profile of P. casabonae, and the identification of sequences specific for this species, enables to identify useful biomolecular markers to distinguish it unequivocally., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Nowadays, there is an increasing demand for fast and reliable plant biomolecular analyses. Conventional methods for the isolation of nucleic acids are time-consuming and require multiple and often non-automatable steps to remove cellular interferences, with consequence that sample preparation is the major bottleneck in the bioanalytical workflow. New opportunities have been created by the use of magnetic ionic liquids (MILs) thanks to their affinity for nucleic acids., Results: In the present study, a MIL-based magnet-assisted dispersive liquid-liquid microextraction (maDLLME) method was optimized for the extraction of genomic DNA from Arabidopsis thaliana (L.) Heynh leaves. MILs containing different metal centers were tested and the extraction method was optimized in terms of MIL volume and extraction time for purified DNA and crude lysates. The proposed approach yielded good extraction efficiency and is compatible with both quantitative analysis through fluorimetric-based detection and qualitative analysis as PCR amplification of multi and single locus genes. The protocol was successfully applied to a set of plant species and tissues., Conclusions: The developed MIL-based maDLLME approach exhibits good enrichment of nucleic acids for extraction of template suitable for targeted PCR; it is very fast, sustainable and potentially automatable thereby representing a powerful tool for screening plants rapidly using DNA-based methods.

The volatile fraction of foods and of plant-origin materials provides functional information on sample-related variables, and gas-phase extractions are ideal approaches for its accurate chemical characterization. However, for gas-phase sampling, the usual procedures adopted to standardize results from solvent extraction methods are not appropriate: headspace (HS) composition depends on the intrinsic physicochemical analyte properties (volatility, polarity, partition coefficient(s)) and matrix effects. Method development, design, and expression of the results are therefore challenging. This review article focuses on volatile vapor-phase quantitation methods (internal standard normalization, standard addition, stable isotope dilution assay, multiple headspace extraction) and their suitability in different applications. Because of the analyte informative role, the different ways of expressing results (normalized chromatographic area, percent normalized chromatographic areas, and absolute concentrations) are discussed and critically evaluated with examples of quality markers in chamomile, process contaminants (furan and 2-methylfuran) in roasted coffee, and key-aroma compounds from high-quality cocoa.

Room-temperature ionic liquids (ILs) have been shown to be successful as stationary phases (SPs) for gas chromatography in several fields of applications because of their unique and tunable selectivity, low vapor pressure and volatility, high thermal stability (over 300 °C), and good chromatographic properties. This study has been focused on two ILs based on a phosphonium cation (trihexyl(tetradecyl)phosphonium, P 66614 ) combined with different anions, previously shown to be suitable as gas chromatography (GC) SPs. In particular, trihexyl(tetradecyl)phosphonium bis[(trifluoromethyl)sulfonyl]imide ([P 66614 + ] [NTf 2 - ]) and trihexyl(tetradecyl)phosphonium chloride ([P 66614 + ] [Cl - ]) were investigated, as the Abraham linear solvation energy relationship has shown their ability to interact with the solute(s) when tested with a set of 26-34 probe analytes. The chromatographic performance were investigated on narrow bore and conventional test columns using the following: i) Grob test, ii) a group of model mixtures of compounds characteristic of the flavor, fragrance and essential oil fields (FFMix), iii) a standard mixture of 29 volatile allergens (AlMix), and iv) two essential oils of different complexity (sage and vetiver essential oils). The columns coated with the investigated IL SPs were characterized by similar polarity (Polarity Number (PN): 37 for [P 66614 + ] [Cl - ] and 33 for [P 66614 + ] [NTf 2 - ]), high efficiency and highly satisfactory inertness. The two IL SPs also exhibited a completely different separation performance, with [P 66614 + ] [Cl - ] test columns mainly characterized by high retention and selectivity based on the analyte functional groups, and [P 66614 + ] [NTf 2 - ] test columns featured by short retention and selectivity mainly related to the analyte volatility and polarity. These results were also confirmed with the analysis of sage and vetiver essential oils., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Aroma is a primary hedonic aspect of a good coffee. Coffee aroma quality is generally defined by cup tasting, which however is time-consuming in terms of panel training and alignment and too subjective. It is challenging to define a relationship between chemical profile and aroma sensory impact, but it might provide an objective evaluation of industrial products. This study aimed to define the chemical signature of coffee sensory notes, to develop prediction models based on analytical measurements for use at the control level. In particular, the sensory profile was linked with the chemical composition defined by HS-SPME-GC-MS, using a chemometric-driven approach. The strategy was found to be discriminative and informative, identifying aroma compounds characteristic of the selected sensory notes. The predictive ability in defining the sensory scores of each aroma note was used as a validation tool for the chemical signatures characterized. The most reliable models were those obtained for woody, bitter, and acidic properties, whose selected volatiles reliably represented the sensory note fingerprints. Prediction models could be exploited in quality control, but compromises must be determined if they are to become complementary to panel tasting.

Fragrances and products deriving from essential oils are often formulated or diluted in aqueous media, usually ethanol/water. Gas chromatography (GC) is the technique of choice to analyze volatiles. However, when using columns coated with conventional stationary phases, its application to aqueous samples often requires time-consuming and/or discriminative sample preparation techniques to extract the target analytes from the aqueous medium, so as to avoid its direct injection. In GC with conventional columns, water produces peak asymmetry, poor sensitivity and efficiency, strong adsorption, stationary phase degradation, and, last but not least, it is not easy to detect reliably when present in high amounts. In 2012, Armstrong's group introduced new fully water-compatible ionic-liquid (IL)-based GC capillary columns based on phosphonium and imidazolium derivative cations combined with trifluoromethanesulphonate. These columns were recently made available commercially by Supelco, under the trade name Watercol™. These derivatives maintain IL's unique selectivity and chromatographic properties, and enable water to be used as injection solvent, thus avoiding the sample preparation procedures required by conventional columns. This study reports and critically discusses the results of commercially available water-compatible IL columns for direct analysis of aqueous samples in the fragrance and essential oil fields by GC with thermal conductivity (TCD) and/or flame ionization detectors (FID). The results showed that water-compatible IL-based stationary phases can successfully be adopted for qualitative and quantitative analysis of fragrances and essential oils directly diluted in aqueous solvents. On the other hand, the study also shows that their inertness needs to be further increased and (possibly) the range of operative temperature extended when water is the main solvent of the sample.

Fungal co-cultivation has emerged as a promising way for activating cryptic biosynthetic pathways and discovering novel antimicrobial metabolites. For the success of such studies, a key element remains the development of standardized co-cultivation methods compatible with high-throughput analytical procedures. To efficiently highlight induction processes, it is crucial to acquire a holistic view of intermicrobial communication at the molecular level. To tackle this issue, a strategy was developed based on the miniaturization of fungal cultures that allows for a concomitant survey of induction phenomena in volatile and non-volatile metabolomes. Fungi were directly grown in vials, and each sample was profiled by head space solid phase microextraction gas chromatography mass spectrometry (HS-SPME-GC-MS), while the corresponding solid culture medium was analyzed by liquid chromatography high resolution mass spectrometry (LC-HRMS) after solvent extraction. This strategy was implemented for the screening of volatile and non-volatile metabolite inductions in an ecologically relevant fungal co-culture of Eutypa lata (Pers.) Tul. & C. Tul. (Diatrypaceae) and Botryosphaeria obtusa (Schwein.) Shoemaker (Botryosphaeriaceae), two wood-decaying fungi interacting in the context of esca disease of grapevine. For a comprehensive evaluation of the results, a multivariate data analysis combining Analysis of Variance and Partial Least Squares approaches, namely AMOPLS, was used to explore the complex LC-HRMS and GC-MS datasets and highlight dynamically induced compounds. A time-series study was carried out over 9 days, showing characteristic metabolite induction patterns in both volatile and non-volatile dimensions. Relevant links between the dynamics of expression of specific metabolite production were observed. In addition, the antifungal activity of 2-nonanone, a metabolite incrementally produced over time in the volatile fraction, was assessed against Eutypa lata and Botryosphaeria obtusa in an adapted bioassay set for volatile compounds. This compound has shown antifungal activity on both fungi and was found to be co-expressed with a known antifungal compound, O -methylmellein, induced in solid media. This strategy could help elucidate microbial inter- and intra-species cross-talk at various levels. Moreover, it supports the study of concerted defense/communication mechanisms for efficiently identifying original antimicrobials.

Objective: The present experiment has tested the effect of dietary alfalfa flavonoids (AAF) supplementation on the productive performances, carcass characteristics, meat quality and lipid oxidation of growing rabbits., Methods: One hundred and sixty crossbred rabbits (42 days old) were divided into four groups of forty animals each and were fed either a control diet (AAF0) or an AAF0 diet supplemented with 400, 800, or 1,200 mg of AAF/kg per diet (AAF4, AAF8, and AAF12, respectively) from weaning to slaughtering (102 days old). Performance data were recorded over a period of 60 days. At the end of the trial, 12 rabbits were slaughtered per group, and the carcass characteristics were recorded. Moreover, the plasma, liver and dorsal muscles were sampled from 12 rabbits/group, and were analyzed for lipid oxidation., Results: No significant differences were recorded for the performance, carcass characteristics and meat quality traits except for lightness parameter that was lower in the control group. Dietary AAF supplementation significantly (p<0.01) affected the malondialdehyde (MDA) levels of the frozen meat in a dose-related manner, with the lowest value (0.24 mg MDA/kg fresh meat) recorded in the AAF12 group samples., Conclusion: These findings indicated that the dietary inclusion of AAF in rabbit diets improved muscle oxidation stability with no adverse effects on the growth performance of the animals even if a slight impact on meat lightness color parameter was recorded.

Ethnopharmacological Relevance: Thistles species (Family: Compositae) are traditionally used in the Mediterranean area, particularly in Sardinia. They are usually gathered from the wild and used for both food and therapeutic purposes, including gastrointestinal disorders., Aim of the Study: This work aims to evaluate the anti-inflammatory activity of eight wild thistles from Sardinia, in an in vitro model of gastric inflammation, and to identify the major active compounds in the extracts., Materials and Methods: The hydro-alcoholic extract of the aerial part of each species was prepared. After the induction of inflammation by the addition of tumor necrosis factor-α (TNFα) (10ng/mL), AGS cells were treated with extracts/pure compounds under study. The inhibition of interleukin-8 (IL-8) release, IL-8 and NF-κB promoter activities and NF-κB nuclear translocation were evaluated. Extracts main components were identified by HPLC-PDA-MS/MS., Results: Only Onopordum horridum Viv. and Onopordum illyricum L. hydro-alcoholic extracts reduced, in a concentration-dependent fashion, the IL-8 release and promoter activity in human gastric epithelial cells AGS. The effect was partially due to the NF-κB pathway impairment. Onopordum hydro-alcoholic extracts were also chemically profiled, and caffeoylquinic acid derivatives were the main compounds identified in the extract. Further investigations showed that 3,5 dicaffeoylquinic acid highly inhibited IL-8 secretion in AGS cells (IC 50 0.65μM), thus suggesting that this compound contributed, at least in part, to the anti-inflammatory activity elicited by O. illyricum extracts., Conclusions: Our results suggest that Onopordum species may exert beneficial effects against gastric inflammatory diseases. Thus, these wild plants deserve further investigations as preventive or co-adjuvant agents in gastric diseases., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Carduus species (Compositae) are widely distributed in the Mediterranean area, and traditionally used for both food and medicinal purposes. The hydroalcoholic extracts of four wild edible Carduus species collected in Sardinia (Carduus argyroa Biv., Carduus nutans subsp. macrocephalus (Desf.) Nyman, Carduus pycnocephalus L., Carduus cephalanthus Viv.) were analyzed and characterized by HPLC-PDA-MS/MS and PCR-RFLP of the nrDNA internal transcribed spacer (ITS). Flavonoids and caffeoylquinic acid derivatives were the predominant classes of secondary metabolites characterizing the extracts. The ITS region was sequenced in parallel, and a PCR-RFLP method was applied with three selective restriction enzymes. Statistical analyses, on both chemical and biomolecular results, revealed that individuals clustered according to their taxonomic classification. The combination of the two techniques discriminates the four species within the genus, giving further information on these little-investigated plants, traditionally used in the Mediterranean area and in Sardinia., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Tea prepared by infusion of dried leaves of Camellia sinensis (L.) Kuntze, is the second world's most popular beverage, after water. Its consumption is associated with its chemical composition: it influences its sensory and nutritional quality addressing consumer preferences, and potential health benefits. This study aims to obtain an informative chemical signature of the volatile fraction of black tea samples from Ceylon by applying the principles of sensomics. In particular, several high concentration capacity (HCC) sample preparation techniques were tested in combination with GC×GC-MS to investigate chemical signatures of black tea volatiles. This platform, using headspace solid phase microextraction (HS-SPME) with multicomponent fiber as sampling technique, recovers 95% of the key-odorants in a fully automated work-flow. A group 123 components, including key-odorants, technological and botanical tracers, were mapped. The resulting 2D fingerprints were interpreted by pattern recognition tools (i.e. template matching fingerprinting and scripting) providing highly informative chemical signatures for quality assessment., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In the fields of essential oils and fragrances, samples often consist of mixtures of compounds with similar structural and physical characteristics (e.g. mono- and sesquiterpenoids), whose correct identification closely depends on the synergic combination of chromatographic and mass spectral data. This sample complexity means that new GC stationary phases with different selectivities are continually being investigated. Ionic liquids (ILs) are of great interest as GC stationary phases in this field because of their selectivity (significantly different than that of currently phases) and their high temperature stability. A first generation of IL GC columns was found to be competitive when applied to these field, in terms of selectivity and efficiency, compared to conventional columns (polydimethylsiloxane, (e.g. OV-1), methyl-polysiloxane 5%-phenyl (e.g. SE-52), 7%-cyanopropyl, 7%-phenyl polysiloxane (e.g. OV-1701), and polyethylen glycol (e.g. PEG-20M). However, these columns showed significant activity towards polar or active analytes, which primarily affected their quantitative performance. A new generation of highly-inactive columns coated with three of the most widely-used ionic liquid GC stationary phases has recently been introduced; these phases are SLB-IL60i (1,12-di(tripropylphosphonium) dodecane bis(trifluoromethylsulfonyl) imide [NTf 2 ], SLB-IL76i (tri-(tripropylphosphonium-hexanamido)-triethylamine [NTf 2 ]), and SLB-IL111i (1,5-di (2,3-dimethyllimidazolium) pentane [NTf 2 ]). This study carefully tested the new inert IL columns, in view of their routine application in the fragrance and essential oil fields. They were found to have unusually high selectivity, comparable to that of first-generation IL columns, while their inertness and efficiency were competitive with those of currently-used conventional columns. The IL column performance of first and second generations was compared, through the quali-quantitative analysis of components in a group of different complexity samples; these included the Grob test, a standard mixture of "suspected" skin allergens, and the essential oils of chamomile and sandalwood., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Artemisia umbelliformis, commonly known as "white génépi", is characterized by a volatile fraction rich in α- and β-thujones, two monoterpenoids; under European Union (EU) regulations these are limited to 35 mg/L in Artemisia-based beverages because of their recognized activity on the human central nervous system. This study reports the results of an investigation to define the geographical origin and thujone content of individual plants of A. umbelliformis from different geographical sites, cultivated experimentally at a single site, and to predict the thujone content in the resulting liqueurs through their volatile fraction. Headspace solid phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS) and non-separative HS-SPME-MS were used as analytical platforms to create a database suitable for chemometric description and prediction through linear discriminant analysis (LDA). HS-SPME-MS was applied to shorten analysis time. With both approaches, a diagnostic prediction of (i) plant geographical origin and (ii) thujone content of plant-related liqueurs could be made.

Recent technological advances in dynamic headspace sampling (D-HS) and the possibility to automate this sampling method have lead to a marked improvement in its the performance, a strong renewal of interest in it, and have extended its fields of application. The introduction of in-parallel and in-series automatic multi-sampling and of new trapping materials, plus the possibility to design an effective sampling process by correctly applying the breakthrough volume theory, have make profiling more representative, and have enhanced selectivity, and flexibility, also offering the possibility of fractionated enrichment in particular for high-volatility compounds. This study deals with fractionated D-HS ability to produce a sample representative of the volatile fraction of solid or liquid matrices. Experiments were carried out on a model equimolar (0.5mM) EtOH/water solution, comprising 16 compounds with different polarities and volatilities, structures ranging from C5 to C15 and vapor pressures from 4.15kPa (2,3-pentandione) to 0.004kPa (t-β-caryophyllene), and on an Arabica roasted coffee powder. Three trapping materials were considered: Tenax TA™ (TX), Polydimethylsiloxane foam (PDMS), and a three-carbon cartridge Carbopack B/Carbopack C/Carbosieve S-III™ (CBS). The influence of several parameters on the design of successful fractionated D-HS sampling. Including the physical and chemical characteristics of analytes and matrix, trapping material, analyte breakthrough, purge gas volumes, and sampling temperature, were investigated. The results show that, by appropriately choosing sampling conditions, fractionated D-HS sampling, based on component volatility, can produce a fast and representative profile of the matrix volatile fraction, with total recoveries comparable to those obtained by full evaporation D-HS for liquid samples, and very high concentration factors for solid samples., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The volatile fraction of murici, bacuri and sapodilla are here studied because of their increasing interest for consumers, abundance of production in Brazil, and the general demand for new flavors and aromas. Their volatile profiles were studied by two High Concentration Capacity Headspace techniques (HCC-HS), Headspace Solid Phase Microextraction (HS-SPME) and Headspace Sorptive Extraction (HSSE), in combination with GC-MS. Murici volatile fraction mainly contains esters (38%), carboxylic acids (19%), aldehydes (11%), alcohols (14%), others (13%) and sulfur compounds; bacuri is characterized by terpenes (41%), non-terpenic alcohols (24%), esters (15%), aldehydes (6%), and others (12%); sapodilla consists of esters (33%), alcohols (27%), terpenes (18%) and others (21%). The GC-MS component co-elution was overcome by GC×GC-qMS. The adoption of modern analysis technologies afforded to achieve a better knowledge of the volatile fraction composition of these fruit pulps by increasing substantially the number of compounds identified., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: A chemical cross-talk between plants and insects is required in order to achieve a successful co-adaptation. In response to herbivory, plants produce specific compounds, and feeding insects respond adequately7 to molecules produced by plants. Here we show the role of the gut microbial community of the mint beetle Chrysolina herbacea in the chemical cross-talk with Mentha aquatica (or watermint)., Results: By using two-dimensional gas chromatography-mass spectrometry we first evaluated the chemical patterns of both M. aquatica leaf and frass volatiles extracted by C. herbacea males and females feeding on plants, and observed marked differences between males and females volatiles. The sex-specific chemical pattern of the frass paralleled with sex-specific distribution of cultivable gut bacteria. Indeed, all isolated gut bacteria from females belonged to either α- or γ-Proteobacteria, whilst those from males were γ-Proteobacteria or Firmicutes. We then demonstrated that five Serratia marcescens strains from females possessed antibacterial activity against bacteria from males belonging to Firmicutes suggesting competition by production of antimicrobial compounds. By in vitro experiments, we lastly showed that the microbial communities from the two sexes were associated to specific metabolic patterns with respect to their ability to biotransform M. aquatica terpenoids, and metabolize them into an array of compounds with possible pheromone activity., Conclusions: Our data suggest that cultivable gut bacteria of Chrysolina herbacea males and females influence the volatile blend of herbivory induced Mentha aquatica volatiles in a sex-specific way.

Salvia desoleana Atzei & V. Picci is an indigenous species in Sardinia island used in folk medicine to treat menstrual, digestive and central nervous system diseases. Nowadays, it is widely cultivated for the pleasant smell of its essential oil (EO), whose antimicrobial and antifungal activities have already been screened. This study evaluated the in vitro anti-Herpes Simplex Virus-2 (HSV-2) activity of S. desoleana EO, fractions and main components: linalyl acetate, alpha terpinyl acetate, and germacrene D. Phytochemical composition of S. desoleana EO was studied by GC-FID/MS analysis and the active fraction(s) and/or compounds in S. desoleana EO were identified with a bioassay-guided fractionation procedure through in vitro assays on cell viability and HSV-2 and RSV inhibition. S. desoleana EO inhibits both acyclovir sensitive and acyclovir resistant HSV-2 strains with EC50 values of 23.72 μg/ml for the former and 28.57 μg/ml for the latter. Moreover, a significant suppression of HSV-2 replication was observed with an EC50 value of 33.01 μg/ml (95% CI: 26.26 to 41.49) when the EO was added post-infection. Among the fractions resulting from flash column chromatography on silica gel, the one containing 54% of germacrene D showed a similar spectrum of activity of S. desoleana EO with a stronger suppression in post-infection stage. These results indicated that S. desoleana EO can be of interest to develop new and alternative anti-HSV-2 products active also against acyclovir-resistant HSV-2 strains.

Background: Human rotavirus (HRoV) is the leading cause of severe gastroenteritis in infants and children under the age of five years. No specific antiviral drug is available for HRoV infections and the treatment of viral diarrhea is mainly based on rehydration and zinc treatment. In this study, we explored medicinal plants endemic to Turkey flora as a source of anti-HRoV compunds., Methods: We performed an antiviral screening on Ballota macrodonta, Salvia cryptantha and Rindera lanata extracts by focus reduction assay. The extract with the highest selectivity index (SI) was selected; its antiviral activity was further confirmed against other HRoV strains and by virus yield reduction assay. The step of viral replicative cycle putatively inhibited was investigated by in vitro assays., Results: The methanolic extract of R. lanata (Boraginaceae) showed the most favourable selectivity index. This extract exhibited a dose-dependent inhibitory activity against three different HRoV strains (EC 50 values ranging from 5.8 μg/ml to 25.5 μg/ml), but was inactive or barely active against other RNA viruses, namely human rhinovirus and respiratory syncytial virus. The R. lanata extract targets the early steps of HRoV infection, likely by hampering virus penetration into the cells., Conclusion: These results make the R. lanata methanolic extract a promising starting material for a bioguided-fractionation aimed at identifying anti-HRoV compounds. Further work is required to isolate the active principle and assess its clinical potential.

The possibility to transfer methods from thermal to differential-flow modulated comprehensive two-dimensional gas chromatographic (GC×GC) platforms is of high interest to improve GC×GC flexibility and increase the compatibility of results from different platforms. The principles of method translation are here applied to an original method, developed for a loop-type thermal modulated GC×GC-MS/FID system, suitable for quali-quantitative screening of suspected fragrance allergens. The analysis conditions were translated to a reverse-injection differential flow modulated platform (GC×2GC-MS/FID) with a dual-parallel secondary column and dual detection. The experimental results, for a model mixture of suspected volatile allergens and for raw fragrance mixtures of different composition, confirmed the feasibility of translating methods by preserving 1 D elution order, as well as the relative alignment of resulting 2D peak patterns. A correct translation produced several benefits including an effective transfer of metadata (compound names, MS fragmentation pattern, response factors) by automatic template transformation and matching from the original/reference method to its translated counterpart. The correct translation provided: (a) 2D pattern repeatability, (b) MS fragmentation pattern reliability for identity confirmation, and (c) comparable response factors and quantitation accuracy within a concentration range of three orders of magnitude. The adoption of a narrow bore (i.e. 0.1mm d c ) first-dimension column to operate under close-to-optimal conditions with the differential-flow modulation GC×GC platform was also advantageous in halving the total analysis under the translated conditions., (Copyright © 2016 Elsevier B.V. All rights reserved.)