Cytotoxicity and Chemotaxonomic Significance of Saponins from Wild and Cultured Asparagus Shoots.

The shoots of Asparagus L. are consumed worldwide, although most species belonging to this genus have a restricted range, and several taxa remain unstudied. In this work, a total of four taxa from different locations were scrutinized and compared with cultivated A. officinalis. All shoots were screened for saponins via LC-MS, and in vitro antiproliferative activities against the HT-29 colorectal cancer cell line were assessed via the MTT assay. The total saponins (TS) contained in the crude extracts ranged from 710.0 (A. officinalis) to 1258.6 mg/100 g dw (A. acutifolius). The richness of the compounds detected in this work stands out; a total of 47 saponins have been detected and quantified in the edible parts (shoots) of five taxa of Asparagus. The structure of all the saponins found present skeletons of the furostane and spirostane type. In turn, the structures with a furostane skeleton are divided into unsaturated and dioxygenated types, both in the 20-22 position. The sum of dioscin and derivatives varied largely among the studied taxa, reaching the following percentages of TS: 27.11 (A. officinalis), 18.96 (A. aphyllus), 5.37 (A. acutifolius), and 0.59 (A. albus); while in A. horridus, this compound remains undetected. Aspachiosde A, D, and M varied largely among samples, while a total of seven aspaspirostanosides were characterized in the analyzed species. The hierarchical cluster analysis of the saponin profiles clearly separated the various taxa and demonstrated that the taxonomic position is more important than the place from which the samples were acquired. Thus, saponin profiles have chemotaxonomic significance in Asparagus taxa. The MTT assay showed dose- and time-dependent inhibitory effects of all saponins extracts on HT-29 cancer cells, and the strongest cell growth inhibition was exercised by A. albus and A. acutifolius (GI50 of 125 and 175 µg/mL). This work constitutes a whole approach to evaluating the saponins from the shoots of different Asparagus taxa and provides arguments for using them as functional foods.

Wild Asparagus Shoots Constitute a Healthy Source of Bioactive Compounds.

Wild Asparagus shoots are consumed worldwide, although most species remain understudied. In this work, a total of four wild Asparagus species were collected from different locations and analyzed compared with farmed A. officinalis. Shoots were screened for (i) phenolic compounds by HPLC-DAD and LC-MS; (ii) total phenolic acids and total flavonoid content by the Folin-Ciocalteu and aluminum chloride methods; (iii) vitamin C by HPLC-DAD; (iv) antioxidant activity by the DPPH and ABTS•+ methods; and (v) the in vitro antiproliferative activities against HT-29 colorectal cancer cells by the MTT assay. Phenolics ranged from 107.5 (A. aphyllus) to 605.4 mg/100 g dry weight (dw) (A. horridus). Vitamin C ranged from 15.8 (A. acutifolius) to 22.7 mg/100 g fresh weight (fw) (A. officinalis). The antioxidant activity was similar in all species, standing out in A. officinalis with 5.94 (DPPH) and 4.64 (ABTS) mmol TE/100 g dw. Among phenolics, rutin reached the highest values (574 mg/100 g dw in A. officinalis), followed by quercetin, nicotiflorin, asterin, and narcissin. The MTT assay revealed the inhibitory effects of ethanol extracts against HT-29 cancer cells, highlighting the cell growth inhibition exercised by A. albus (300 µg/mL after 72 h exposure to cells). This work improves knowledge on the phytochemicals and bioactivities of the shoots of wild Asparagus species and confirms their suitability for use as functional foods.

Assessing differences in the bioaccessibility of phenolics present in two wine by-products using an in-vitro model of fish digestion.

Increasing attention is currently being paid to the protective role of polyphenols in health and oxidative status in fish. For this reason, the potential use of different natural sources of such compounds, like wine by products, is under study. One key step required to gain a better understanding on the biological roles of polyphenols for a given species is to assess the different factors affecting their digestive bioaccessibility, and a great number of such studies is based in the use of in vitro digestion models. In the present study the potential digestive bioavailability of the phenolic compounds present in wine bagasse and lees was evaluated for two fish species showing great differences in their digestive phisyiology: the omnivorous gilthead sea bream (Sparus aurata) and the herbivorous flathead grey mullet (Mugil cephalus). The study was developed using in vitro models adapted to simulate their digestion and a factorial experimental design that simultaneously evaluated the effects of the ingredient used as source of polyphenols, presence or absence of feed matrix, fish species and digestion time. The release of the phenolic compounds was evaluated using ultra-high performance liquid chromatography (UHPLC) coupled to high resolution mass spectrometry (HRMS) detection. Both the presence of feed matrix and the type of wine by-product showed a significant effect on the digestive release of both total and specific types of polyphenols while fish species showed to be significant only for some specific compounds, like eriodyctiol or syringic acid. The time of digestion was not identified as a statistically significant factor in the release of phenolic compounds due to the great variability in the patterns observed that were classified as early, sustained and late. The observed great variations in the patterns of release of different types of phenolic compounds with time suggest an important effect of gut transit rates on the net bioavailability of a given phenolic compound in the live fish. The present study is, to our knowledge, the first one on which an in vitro approach was applied to assess to what extent the possible complexation of wine polyphenols present in wine by-products with either digestive enzymes or components of the feed matrix could limit their bioaccessibility if included in diets of two different fish species.

Arecaceae Seeds Constitute a Healthy Source of Fatty Acids and Phenolic Compounds.

Seeds of most Arecaceae species are an underutilized raw material that can constitute a source of nutritionally relevant compounds. In this work, seeds of 24 Arecaceae taxa were analyzed for fatty acids (FAs) by GC-FID, for phenolics by HPLC-DAD and LC-MS, and for their antitumor activity against the HT-29 colorectal cancer cell line by the MTT assay. Lauric, oleic, and linoleic acids were the prominent FAs. Cocoseae species contained total FAs at 28.0-68.3 g/100 g seeds, and in other species total FAs were from 1.2 (Livistona saribus) to 9.9 g/100 g (Washingtonia robusta). Sabal domingensis, Chamaerops humilis, and Phoenix dactylifera var. Medjool had unsaturated/saturated FA ratios of 1.65, 1.33-1.78, and 1.31, respectively, and contained 7.4, 5.5-6.3, and 6.4 g FAs/100 g seeds, respectively. Thus, they could be used as raw materials for healthy oilseed production. Phenolics ranged between 39 (Livistona fulva) and 246 mg/100 g (Sabal palmetto), and of these, caffeic acid, catechin, dactylifric acid, and rutin had the highest values. (-)-Epicatechin was identified in most seed extracts by LC-MS. Hydroalcoholic extracts from five species showed a dose-dependent inhibitory effect on HT-20 cells growth at 72 h (GI50 at 1533-1968 µg/mL). Overall, Arecaceae seeds could be considered as a cheap source of health-promoting compounds.

Non-targeted analysis of co-formulants in antifungal pesticide formulations by gas chromatography-tandem high resolution mass spectrometry.

In the present study, six commercial pesticide formulations with antifungal activity were characterized. Thus, two complementary injection methods based on gas chromatography were employed: direct injection (DI) and headspace (HS), both coupled to high resolution mass spectrometry (GC-Q-Orbitrap-MS). The combination of both injection modes allowed the tentatively identification of potential co-formulants. Available analytical standards were acquired for their confirmation, and 21 compounds were successfully confirmed. Finally, the concentration of these co-formulants was calculated, finding the highest value in one of the pesticide formulation, at 218.22 g L-1 for cyclohexanone. Results clearly show that this methodology is suitable for the reliable identification of co-formulants in pesticide formulations, offering high sensitivity, and highlighting that five co-formulants were detected by both injection techniques (DI and HS). Moreover, one of the main advantages of the proposed methods was the great capacity for the elucidation of compounds with similar molecular formula, bearing in mind that up to 8 co-formulants with the same molecular formula C10H14, were well differentiated by retention times between 8.46 (1-methyl-3-propylbenzene) and 10.98 min (1,2,3,4- tetramethylbenzene) in one of the pesticide formulation. Toxicity to human health and the environment has been evidenced for the co-formulants detected, finding compounds with relatively high toxicity, as naphthalene and cyclohexanone.

Buglossoides spp. seeds, a land source of health-promoting n-3 PUFA and phenolic compounds.

Ahiflower oil© is extracted from the seeds of Buglossoides arvensis, which contains high amounts of stearidonic acid (SDA, 18:4n-3), while its phenolic composition still is unreported. Moreover, several Buglossoides taxa remain unstudied and could become natural sources of SDA. In this work, seeds of several Buglossoides taxa and Ahiflower oil© were screened for fatty acids, phenolic compounds, and in vitro antiproliferative activities against colorectal cancer cells. Four flavonoids and 16 phenolic acids were identified and quantified. Among Buglossoides taxa, the highest amounts of phenolic compounds were found in samples collected in Spain, under a warm Mediterranean climate. Rosmarinic and lithospermic acids were the main phenols found in Buglossoides seeds. The MTT assay showed dose- and time-dependent inhibitory effects of B. arvensis extracts on HT-29 cancer cells, with a GI50value of ∼280 µg/mL after 72 h of cell exposure to seed extracts. The latter showed lower antiproliferative activity than that of pure phenolics due to the simultaneous presence of other compounds in the extracts, as evidenced by 1H NMR. This work constitutes the first approach to evaluate the seeds of several Buglossoides taxa as functional oils-providers to use them as functional foods.

Co-formulants in plant protection products: An analytical approach to their determination by gas chromatography-high resolution mass accuracy spectrometry.

In the present study, 12 volatile benzene and naphthalene derived co-formulants were identified by suspect screening and unknown analysis in 14 plant protection products (PPPs) corresponding to several types of formulations, as emulsifiable concentrates (EC), suspension concentrates (SC), dispersible concentrates (DC) and ZC, which is a mixture of a capsule suspension (CS) in an SC, containing either difenoconazole or chlorantraniliprole as main active ingredients. The selected technique was gas chromatography coupled to Q-Orbitrap high resolution mass accuracy spectrometry (GC-Q-Orbitrap-MS), providing efficient separation and detection of all identified compounds. Finally, 42 compounds were tentatively identified, and 12 of them were confirmed and quantified using analytical standards. Results showed that the applied methodology was able to detect these co-formulants at concentrations as low as 0.03 g/L (tert-butylbenzene), encompassing a wide concentration range, up to 9.63 g/L (pentamethylbenzene). Pentamethylbenzene was the only compound detected in all studied samples.

Simultaneous determination of polar pesticides in human blood serum by liquid chromatography coupled to triple quadrupole mass spectrometer.

In this study a simple and rapid method was used for the determination of 5 polar pesticides and 3 metabolites, ethephon, 2-hydroxyethyl phosphonic acid (HEPA), fosetyl-aluminum, phosphonic acid, glyphosate, chlorate, glufosinate and 3-methylphosphinicopropionic acid (MPPA) in human blood serum. Acidified water (1% formic acid) and acetonitrile (1% formic acid) were used as extraction solvents and n-hexane was utilized as a clean-up step for lipid removal. The analytical method was based on liquid chromatography coupled to triple quadrupole tandem mass spectrometry (LC-QqQ-MS/MS). The proposed method was validated, obtaining recoveries ranging from 70 to 110% and precision values lower than 17% for all analytes. Limit of quantification was 0.01 mg/L for ethephon, fosetyl-aluminum, MPPA and HEPA, 0.025 mg/L for glyphosate, phosphonic acid and chlorate and 0.05 mg/L for glufosinate. This is the first time that an analytical method was developed for the determination of a high number of polar pesticides in human blood serum, allowing the simultaneous monitoring of these compounds in a complex matrix as human blood serum.

A new strategy based on gas chromatography-high resolution mass spectrometry (GC-HRMS-Q-Orbitrap) for the determination of alkenylbenzenes in pepper and its varieties.

Alkenylbenzenes are natural toxins with genotoxic and carcinogenic effects in rodents, which are highly present in condiments frequently consumed. The aim of this study was the development of the first multi-analyte method for the determination of eight alkenylbenzenes (eugenol, methyl eugenol, acetyl eugenol, trans-isoeugenol, safrole, estragole, myristicin and trans-anethole) in different pepper varieties by gas chromatography coupled to high-resolution mass spectrometry (GC-HRMS-Q-Orbitrap) in combination with a simple ultrasound-assisted extraction method (UAE). The method was successfully validated, and it was applied for studying the presence of these analytes in peppers as well as to elucidate the effects of the berries' maturity and the geographical origin on alkenylbenzene contents. The analysis of the pepper samples showed that eugenol (10.5-120 mg/kg), trans-anethole (10.7-42.7 mg/kg) and estragole (2.2-45.7 mg/kg) tended to be the most detected alkenylbenzenes at high levels, whereas trans-isoeugenol (0.69-3.6 mg/kg) and safrole (0.20-3.0 mg/kg) were minor components. Estragole (PubChem CID: 8815); trans-anethole (PubChem CID: 637563); Myristicin (PubChem CID: 4276); Safrole (PubChem CID: 5144); Eugenol (PubChem CID: 3314); Methyl eugenol (PubChem CID: 7127); Acetyl eugenol (PubChem CID: 7136); trans-Isoeugenol (PubChem CID: 853433); Caffeine (PubChem CID: 2519); Dicyclohexylmethanol (PubChem CID: 78197).

Dissipation kinetic studies of fenamidone and propamocarb in vegetables under greenhouse conditions using liquid and gas chromatography coupled to high-resolution mass spectrometry.

In this study, fenamidone, propamocarb and their transformation products were monitored in cherry tomato, cucumber, and courgette samples. A mixture of both compounds, which have different physico-chemical characteristics, are commercially available (Consento®). For analysis, ultra high-performance liquid chromatography coupled to Orbitrap mass spectrometry (UHPLC-Orbitrap-MS) and gas chromatography coupled to Q-Orbitrap mass spectrometry (GC-Q-Orbitrap-MS) were used. The dissipation of these active ingredients was monitored at two doses (normal and double dose) from 1 to 40 days after the application of the commercial product. Half-lives (DT50) were lower than 30 days for both compounds, which indicates low persistence. Metabolites of both compounds were also monitored due to in some cases these can be more dangerous for human health than the parent compounds. The metabolites monitored were RPA 410193 ((5S)-3-anilino-5-methyl-5-phenylimidazolidine-2,4-dione), acetophenone, 2-phenylpropionic acid, 5-methyl-5-phenylhydantoin and 5-methylhydantoin for fenamidone, and propamocarb hydrochloride (propyl 3-(dimethylamino)propylcarbamate hydrochloride), N-oxide propamocarb (propyl [3-(dimethylnitroryl)propyl]carbamate), oxazoline-2-one propamocarb (3-[3-(dimethylamino)propyl]-4-hydroxy-4-methyl-1,3-oxazolidin-2-one), 2-hydroxypropamocarb and n-desmethyl propamocarb (propyl [3-(methylamino)propyl]carbamate) for propamocarb. In addition, they were detected one day after the application of commercial product, being RPA 410193, the metabolite detected at the highest concentration in samples. Retrospective analysis of incurred samples allowed putative identification of four possible new metabolites of propamocarb and one of fenamidone.

A new anthraquinoid ligand for the iron-catalyzed hydrosilylation of carbonyl compounds at room temperature: new insights and kinetics.

The reaction of 1-((2-(pyridin-2-yl)ethyl)amino)anthraquinone with either Fe(HMDS)2 or Li(HMDS)/FeCl2 allowed the preparation of a new anthraquinoid-based iron(ii) complex active in the hydrosilylations of carbonyls. The new complex Fe(2)2 was characterized by single-crystal X-ray diffraction, infrared spectroscopy, NMR, and high resolution mass spectrometry (electrospray ionization). Superconducting quantum interference device (SQUID) magnetometry established no spin crossover behavior with an S = 2 state at room temperature. This complex was determined to be an effective catalyst for the hydrosilylation of aldehydes and ketones, exhibiting turnover frequencies of up to 63 min-1 with a broad functional group tolerance by just using 0.25 mol% of the catalyst at room temperature, and even under solvent-free conditions. The aldehyde hydrosilylation makes it one of the most efficient first-row transition metal catalysts for this transformation. Kinetic studies have proven first-order dependences with respect to acetophenone and Ph2SiH2 and a fractional order in the case of the catalyst.

Headspace solid-phase microextraction coupled to gas chromatography-tandem mass spectrometry for the determination of haloanisoles in sparkling (cava and cider) and non-sparkling (wine) alcoholic beverages.

A highly sensitive analytical method was developed to determine 2,4,6-trichloroanisole (TCA), 2,3,4,6-tetrachloroanisole (TeCA), 2,4,6-tribromoanisole (TBA) and 2,3,4,5,6-pentachloroanisole (PCA) in sparkling alcoholic beverages. The method was based on the use of headspace solid-phase microextraction (HS-SPME) using a polydimethylsiloxane (PDMS) fibre. It was coupled to gas chromatography-triple quadrupole tandem mass spectrometry (GC-QqQ-MS/MS) for the detection and quantification of the target haloanisoles. The method was fully automated and no sample preparation was needed. The method was validated for alcoholic beverages. The influence of CO2 on the extraction efficiency was also evaluated for the studied sparkling drinks (cava and cider). All the calibration curves showed good linearity (R2 > 0.98) within the tested range (1-50 ng l-1). Recoveries were evaluated at three different levels (1, 5 and 50 ng l-1) and were always between 71% and 119%. Precision was expressed as relative standard deviation (RSD), and was evaluated as intra- and inter-day precisions, with values ≤ 22% in both cases. Limits of quantitation (LOQs) were ≤ 0.91 ng l-1, which are below the sensory threshold levels for such compounds in humans. The validated method was applied to commercial samples, 10 cavas and 10 ciders, but it was also used for the analysis of nine red wines and four white wines, demonstrating the further applicability of the proposed method to non-sparkling beverages. TCA was detected in most samples at up to 0.45 ng l-1.

Determination of polycyclic aromatic hydrocarbons in soy isoflavone nutraceutical products by gas chromatography coupled to triple quadrupole tandem mass spectrometry.

Thirteen polycyclic aromatic hydrocarbons have been determined in soy-based nutraceutical products. First, an optimization of extraction procedure was performed, and a solid-liquid extraction assisted by sonication and a dilute and shoot procedure were compared, selecting the dilute and shoot approach for the extraction of target compounds, utilizing a mixture of acetone/n-hexane (1:1 v/v) as extractant solvent. After this, a clean-up step was needed bearing in mind the complexity of these matrices. Dispersive solid-phase extraction, using a mixture of C18 and Zr-Sep+ (25 mg/mL each) was used. The separation was achieved by gas chromatography and detection with triple quadrupole tandem mass spectrometry. For quantification purposes, matrix-matched calibration was used. The validation was applied at three concentration levels (20, 100 and 250 µg/kg), obtaining recoveries between 70 and 120% and precision values equal to or lower than 23%. Limits of detection and quantification were below 8 and 20 µg/kg, respectively. The method was applied in 11 samples, detecting five polycyclic aromatic hydrocarbons at concentrations ranging from 4.1 to 18.5 µg/kg.