Agronomic, Nutritional Traits, and Alkaloids of Lupinus albus, Lupinus angustifolius and Lupinus luteus Genotypes: Effect of Sowing Dates and Locations.

Lupins (Lupinus spp.) are legumes with high relevance for the sustainability of agricultural systems as they improve the soil quality, namely, through the fixation of atmospheric nitrogen, and have good adaptability to different climates and soil conditions. Besides, they possess high nutritive value, especially due to the high protein content of the seeds. Nevertheless, the plants' productivity and metabolism can be influenced by the genotype, the edaphoclimatic conditions, and the sowing practices. In this work, the effect of edaphoclimatic conditions and sowing dates on the productivity, nutritional factors, and alkaloids of the seeds of L. albus cv. Estoril, L. angustifolius cv. Tango, and L. luteus cv. Cardiga was evaluated. High variability in the seeds and protein productions, nutritional traits, and alkaloid content related to the species was observed, along with a significant effect of the location. Lupinus albus cv. Estoril showed a good compromise between productivity and low alkaloid content, being an interesting genotype for food and feed use in the conditions of this trial.

Environmental conditions affect the nutritive value and alkaloid profiles of Lupinus forage: Opportunities and threats for sustainable ruminant systems.

The identification of crops that simultaneously contribute to the global protein supply and mitigate the effects of climate change is an urgent matter. Lupins are well adapted to nutrient-poor or contaminated soils, tolerate various abiotic stresses, and present relevant traits for acting as ecosystem engineers. Lupins are best studied for their seeds, but their full foraging potential needs further evaluation. This study evaluated the effects of location and sowing date on forage production, proximate composition, and the detailed mineral and alkaloid profiles of three species of Lupinus (L. albus cv. Estoril, L. angustifolius cv. Tango, and L. luteus cv. Cardiga). Sowing date and location and their interaction with the plant species significantly affected the vast majority of measured parameters, emphasizing the effects of climate and soil conditions on these crops. The relatively high crude protein and in vitro digestibility support the potential of the lupin species studied as sustainable forage protein sources in diets for ruminant animals. The content of individual essential macro and trace elements was below the maximum tolerable levels for cattle and sheep. Lupanine, smipine, and sparteine were the most abundant quinolizidine alkaloids in L. albus cv. Estoril, lupanine, and sparteine in L. angustifolius cv. Tango, and lupinine, gramine, ammodendrine, and sparteine in L. luteus cv. Cardiga. Based on the maximum tolerable levels of total quinolizidine alkaloid intake, the dietary inclusion of forages of L. albus cv. Estoril and L. angustifolius cv. Tango does not pose a risk to the animals, but the high alkaloid content of L. luteus cv. Cardiga may compromise its utilization at high levels in the diet. Overall, the results reveal a high potential for lupins as protein forage sources well adapted to temperate regions and soils with lower fertility, with a relevant impact on livestock sustainability in a climate change era.

Ruminal inocula with distinct fermentation profiles differentially affect the in vitro fermentation pattern of a commercial algal blend.

The in vitro rumen batch technique is widely used for screening novel feed sources; however, it remains unclear to what extent the in vitro fermentability of non-conventional feed sources is affected by non-adapted ruminal inocula. Thus, in this study, we evaluated the effects of distinct ruminal inocula on the in vitro fermentation parameters of a sustainable non-conventional feed, a commercially available algal blend composed of microalgae (Chlorella vulgaris and Nannochloropsis oceanica) and seaweeds (Ulva sp. and Gracilaria gracilis). First, four late-lactation Holstein cows were fed four forage-based diets varying only in the proportions of basal forage (100% corn silage, 70% corn silage and 30% haylage, 30% corn silage and 70% haylage, and 100% haylage) in a 4 × 4 Latin square design with the last square omitted. After 3 weeks of adaptation, haylage-based diets resulted in ruminal fermentation parameters distinct from those promoted by corn silage-based diets, as reflected in increased pH, ammonia-N contents, and acetate proportions. Individual ruminal fluids derived from each of the four diets were further used as inocula in in vitro incubations. Here, a 1:1 mixture of corn silage and haylage was supplemented with 0, 5, 10, or 15% algal blend and incubated with each inoculum for 24 h in a 4 × 4 factorial design. Total gas and methane production decreased with inocula from cows fed haylage-based diets and with increasing algal blend supplementation levels. The fermentation pH increased and the ammonia-N contents decreased with inocula from cows fed haylage-based diets; however, these parameters were not affected by algal blend inclusion levels. The interaction between the ruminal inoculum source and the algal blend supplementation level affected the total volatile fatty acids (VFA) and the proportions of most individual VFA. Total VFA production decreased with increasing algal supplementation levels, particularly with inocula from cows fed 30% corn silage and 70% haylage; the acetate, propionate, and valerate proportions were only affected by algal blend levels under incubation with 100% corn silage inocula. Overall, our findings highlight the importance of the ruminal inoculum source when assessing the fermentability of non-conventional feed as well as the potential of the algal blend as a natural modulator of ruminal fermentation.

Unravelling the combined impacts of drought and Cu in barley plants - double trouble?

The occurrence of drought in soils, particularly in those contaminated by metals, poses a current threat to crops, as these factors can interact and induce unique stress responses. Therefore, this study mainly focused on understanding the crosstalk between drought and copper (Cu) stress in the physiology of the barley (Hordeum vulgare L.) plant. Using a bifactorial experimental design, seedlings were grown in a natural soil under the following treatments: plants continuously irrigated in uncontaminated soil for 14 days (control); plants continuously irrigated in Cu-contaminated soil (115 mg Cu kg-1) for 14 days (Cu); plants only irrigated during the initials 7 days of growth in uncontaminated soil (drought); plants co-exposed to Cu and drought (combined). After 14 days of growth, the results revealed that drought prevented Cu bioaccumulation in barley roots, which were still severely affected by the metal, both individually and in combination with the water deficit. Furthermore, individual and combined exposure to these stressors resulted in impaired photosynthetic performance in barley plants. Despite the increased activation of enzymatic and non-enzymatic antioxidant defence mechanisms, particularly in the green organs, the plants co-exposed to both stress factors still showed higher oxidative damage, severely impacting biomass production.

Simultaneous determination of carbonyl compounds related to thermal treatment and oxidative stability of infant formulas by gas-diffusion microextraction and high-performance liquid chromatography with ultraviolet detection.

Infant formulae are the only possible alternative to breastfeeding during the first year of life, so it is crucial to assure their innocuousness. Infant formula undergoes heat treatments to ensure safety and shelf life. However, such processes impact health as they lead to the formation of malondialdehyde, acrolein, and α-dicarbonyl compounds, related to Maillard reaction. Thus, there is a need for improved analytical methods to ensure the safety, quality, and nutritional value of infant formulae, and also exploring the potential of specific compounds as indicators for quality control and monitoring purposes. We developed and validated a novel, efficient, and cost-effective method using gas-diffusion microextraction for the simultaneous quantification of carbonyl compounds in infant formula. Malondialdehyde, acrolein, glyoxal, methylglyoxal, and diacetyl were detected as o-phenylenediamine derivatives using HPLC with UV detection. Parameters influencing extraction efficiency were studied using an asymmetric screening design. The validated method has shown excellent linearity, sensitivity, accuracy, and precision. It was applied to analyze 26 infant formula samples, including starter, follow-up, and special formulated powdered infant formula. Methylglyoxal was found in all samples (0.201-3.153 µg mL-1), while malondialdehyde was present only in certain starter formulas (1.033-1.802 µg mL-1). Acrolein (0.510-3.246 µg mL-1), glyoxal (0.109-1.253 µg mL-1), and diacetyl (0.119-2.001 µg mL-1) were detected in various sample types. Principal components and hierarchical cluster analyses have showcased distinct sample clustering based on analyte contents. This study presents a novel methodology for the analysis of markers of thermal treatment and oxidative stability in infant formula. It contributes to the characterization of the products' composition and quality control of infant formulae, thereby enhancing their safety and nutritional adequacy. This study also presents the first reported quantification of acrolein in infant formula and introduces the application of the acrolein-o-phenylenediamine derivative for food analysis.

Upcycling post-harvest biomass residues from native European Lupinus species: from straws and pod shells production to nutritive value and alkaloids content for ruminant animals.

The production of Lupinus seeds for food and feed is increasing worldwide, which results in large amounts of post-harvest biomass residues, considered of low value and left in the field to be burned or incorporated in the soil. To valorize these agricultural wastes, this work aimed to assess their potential as an alternative feed for ruminants. Thus, the production yield, nutritive value, and alkaloid content of straws and pod shells from three native European Lupinus species, L. albus 'Estoril' (white), L. angustifolius 'Tango' (narrow-leafed), and L. luteus 'Cardiga' (yellow), cultivated in two locations, were evaluated. The dry matter (DM) yield of straws and pod shells were the highest for L. albus 'Estoril' (4.10 t ha-1) and the lowest for L. angustifolius 'Tango' (1.78 t ha-1), suggesting a poor adaptation of narrow-leafed lupin to the particularly dry and warm agronomic year. Despite species-specific differences, lupin biomass residues presented higher crude protein (53.0-68.9 g kg-1 DM) and lignin (103-111 g kg-1 DM) content than cereal straws usually used in ruminant feeding, thus resulting in higher metabolizable energy (6.43-6.58 MJ kg-1 DM) content. In vitro digestibility was similar among lupin species (47.7-50.6%) and higher in pod shells (53.7%) than in straws (44.6%). Lupinus albus 'Estoril' and L. luteus 'Cardiga' presented considerable amounts of alkaloids in straws (23.9 and 119 mg kg-1 DM) and pod shells (20.5 and 298 mg kg-1 DM), while no alkaloids were detected in L. angustifolius 'Tango' biomass residues. Considering the combined production of straw and pod shells per lupin species, it is anticipated that lupin biomass residues produced per ha can fulfill 85% of the energy and nearly 50% of protein requirements of a flock of 4 to 9 dry and mid-pregnancy sheep with 50 kg body weight for one year. No negative effects on small (ovine) and large (bovine) ruminant species due to alkaloids are expected, even if biomass residues are consumed at up to 85% DM intake. The large production yield along with its nutritive value unveils the potential of lupin biomass residues valorization as alternative fodder for ruminants, promoting sustainability under a circular economy approach.

Insights from the yield, protein production, and detailed alkaloid composition of white (Lupinus albus), narrow-leafed (Lupinus angustifolius), and yellow (Lupinus luteus) lupin cultivars in the Mediterranean region.

Introduction: Lupins and other legumes have been considered as alternative plant-based protein sources to soybeans for both humans and livestock. Furthermore, they can contribute to more sustainable agricultural systems. The productivity and chemical composition of legumes is highly variable between species, cultivars, and with the edaphoclimatic conditions. Methods: This work evaluated the adaptability of seven Lupinus cultivars in two different sowing locations, during two consecutive years, through the characterization of their seed, as a means of investigating their suitability to be used as a source of food and/or feed. Results and discussion: Lupinus angustifolius cv. Tango and Lupinus luteus cv. Acos were the most stable genotypes across the environments when considering the seed and protein production, while L. luteus cv. Alburquerque and L. luteus cv. Mister showed less variation in the total alkaloid content across the environments. The edaphoclimatic conditions affected seed and protein yields, as higher rainfall resulted in high productivity. The lower temperatures observed in the first year at both locations caused a reduction in the production of alkaloids in L. luteus cv. Acos and Cardiga. Due to the high alkaloid content of some of the studied cultivars their use as food or feed can pose some safety concerns. However, these cultivars can have high levels of resistance to herbivore and insect attacks, which can be of the utmost importance for the use of these crops for recovering poor or exhausted soils.

Untargeted metabolomic profiling of fresh and dried leaf extracts of young and mature Eucalyptus globulus trees indicates differences in the presence of specialized metabolites.

Aqueous extracts from Eucalyptus globulus leaves contain a wide variety of specialized metabolites, mainly polyphenols and appreciable amounts of volatile compounds, which are responsible for their diverse biological activities, such as antioxidant, antimicrobial, and allelopathic features. For this reason, several studies have been conducted to explore the composition of E. globulus leaf extracts for multiple therapeutic and commercial applications. However, so far, the available bibliographic reports only refer to the chemical composition of extracts prepared with leaves from mature trees, leaving much to clarify about the composition of juvenile eucalyptus leaf extracts. Furthermore, there is no consensus regarding the type of leaves, fresh or dried ones, to be used in the extraction procedure, considering the highest recovery of biologically active compounds. In this sense, this study aimed to characterize the chemical composition of aqueous extracts prepared with fresh and dried leaves from young and mature E. globulus trees. For this, leaf biomass from young and mature E. globulus trees was collected in three distinct places from a forest area, and after oven-drying a portion of the leaves, an extraction in hot water was carried out, followed by GC-MS and HPLC-MS/MS analyses. The results revealed that the maturity of eucalyptus trees and biomass drying significantly influenced the volatile and non-volatile composition of the aqueous extracts. Accordingly, while fresh leaf extracts of young trees had great levels of hydrolysable tannins, extracts prepared with fresh leaves from mature trees presented a wide range of terpenes. When dried leaf material was used, extracts had notorious contents of amino acids derivatives, C13 norisoprenoids, fatty and other organic acids. Overall, this study showed, for the first time, that plant maturity (young vs mature) and pre-processing (fresh vs dried) of foliar biomass of E. globulus trees need to be considered in the preparation of leaf aqueous extracts depending on the desired purposes, since major changes in what regards biologically active compounds were found.

Nutritional Composition and Untargeted Metabolomics Reveal the Potential of Tetradesmus obliquus, Chlorella vulgaris and Nannochloropsis oceanica as Valuable Nutrient Sources for Dogs.

The growing pet population is questioning the sustainability of the pet food system. Although microalgae may constitute a more sustainable food resource, the assessment of their potential for canine diets is almost non-existent. The present study aimed to evaluate the potential of three microalgae species (Tetradesmus obliquus, Chlorella vulgaris and Nannochloropsis oceanica) grown locally in industrial photobioreactors as alternative food resources for dogs. A detailed characterization of their nutritional composition and metabolomic profile was carried out and related to the nutritional requirements of dogs. Overall, the essential amino acid content exceeded the amounts required for dogs at all life stages, except methionine and cysteine. The three microalgae were deficient in linoleic acid, N. oceanica presented a linolenic acid content below requirements and T. obliquus and C. vulgaris were deficient in arachidonic and eicosapentaenoic acids. The fiber was mainly composed of insoluble dietary fiber. The mineral profile varied greatly with the microalgae species, demonstrating their different potential for dog feeding. Untargeted metabolomics highlighted glycolipids, glycerolipids and phospholipids as the most discriminating compounds between microalgae species. Overall, the results support the potential of T. obliquus, C. vulgaris and N. oceanica as valuable macro- and micro-nutrients sources for dog feeding.

A Novel Approach for Monitoring the Volatile Metabolome in Biological Samples from Ruminants through Miniaturized Liquid-Liquid Extraction and Multiclass Gas Chromatography Analysis.

A straightforward and versatile methodology for the extraction of volatile metabolites in biological samples from ruminants for gas chromatography analysis is proposed. The methodology was applied in the determination of multiclass metabolites (short-chain fatty acids, aldehydes, alcohols, ketones, esters, phenols, and sulfides) in different analytical matrices (rumen fluid, urine, and feces) collected from Holstein cows. The 24 multiclass volatile metabolites reported in the different biological samples and their respective concentrations were critically discussed in the context of digestive physiology. Most detected compounds are derived from the rumen and lower gut fermentation of carbohydrates, proteins, and lipids or their metabolism, being consistent with the prior state of the art. The proposed method also takes advantage of the already existing tools in animal nutrition laboratories, providing a novel methodological ground that can generate relevant bioanalytical information with a significant impact on ruminant's nutritional studies.

Herbicidal Effects and Cellular Targets of Aqueous Extracts from Young Eucalyptus globulus Labill. Leaves.

Eucalyptus globulus Labill. is a widespread exotic species that contributes to the formation of fire-prone environments, a great concern under climate change conditions. Therefore, sustainable practices to help locals managing eucalyptus stands are needed. In this perspective, harnessing eucalyptus' specialized metabolism as a source of allelochemicals can be a promising approach for weed control. Thus, the main goals of this work were to evaluate the herbicidal potential of post-fire regenerated E. globulus leaves against Portulaca oleracea L. and to unravel the physiological mechanisms behind this phytotoxic action. For this, aqueous extracts of fresh (FLE; 617 g FW L-1) or oven-dried leaves (DLE; 250 g DW L-1) were foliar-sprayed at different dilutions in purslane seedlings. After five weeks, results revealed that DLE at the highest dose detained the greatest herbicidal activity, affecting purslane growth and cellular viability. Moreover, biochemical data pointed towards an overproduction of reactive oxygen species, causing harsh oxidative damage in roots, where the upregulation of important cellular players, like sugars, amino acids, and proline, was not able to reestablish redox homeostasis. Overall, this study proved that dried leaves from young E. globulus had potent herbicidal properties against P. oleracea and can represent a feasible strategy for weed management.

Response of Solanum lycopersicum L. to diclofenac - Impacts on the plant's antioxidant mechanisms.

One emerging problem that recently has become a vastly acknowledged topic of concern is the environmental contamination by pharmaceuticals. Diclofenac (DCF) is one of the most common pharmaceuticals found, due to its high utilization and low removal rate in wastewater treatment processes. In this work, Solanum lycopersicum L. was used as a model to unravel how DCF contamination can affect crops, focusing on the internal mechanisms triggered by this exposure. For this purpose, plants were exposed to two different DCF concentrations (0.5 mg L-1 and 5 mg L-1). Results obtained here point towards a loss of shoot performance when plants were exposed to very high concentrations of DCF, but no delay or loss of yield in the flowering and fruit stages were ascribed to DCF contamination. Our data shows that a state of oxidative stress due to high reactive oxygen species accumulation was associated with this contamination, with very high DCF levels leading to a rise of lipid peroxidation, possibly accentuated by the inhibition of ROS-scavenging enzymes and unable to be counteracted by the visible upregulation of proline and the thiol-based redox network. Overall, these results allow to infer that in the current environmental context, no noticeable negative effects should be associated with the presence of DCF in soils where this crop is cultivated. However, the oxidative stress and lower biomass associated with the highest concentration are alarming, since DCF levels in the environment are continuously increasing and further measures are necessary to assess this problematic.

Unravelling the phytonutrients and antioxidant properties of European Vicia faba L. seeds.

For the first time, seven European varieties of Vicia faba L. seeds were investigated for (1) their phytonutrients profile by HPLC-DAD-MS/MS, (2) total phenolic content (TPC), and (3) antioxidant capacity (DPPH and FRAP assays). A wide range of compounds were identified, namely alkaloids, organic acids, terpenoids, jasmonates, and phenolics; these two latter being the most abundant. TPC ranged between 2.62 and 4.3 mg (gallic acid equivalent) g-1 dry weight, for V. faba major variety Belshi and V. faba minor variety Bauska, respectively. The DPPH radical scavenging capacity showed poor correlation (r = 0.550, P = .041) with TPC, suggesting the presence of other antioxidant sources than phenolics. Still, FRAP was positively correlated with TPC (r = 0.709, P < .01) and DPPH (r = 0.819, P < .01). These results elucidated the phytonutrients and antioxidant properties of V. faba L. seeds as functional food sources.

Profiling of phenolic compounds and antioxidant properties of European varieties and cultivars of Vicia faba L. pods.

Vicia faba L. pods are a by-product generated from the industrial processing of beans for human and animal consumption. As phenolic compounds may play important roles in health, the present work envisaged the phenolic characterization of seven European varieties and cultivars of V. faba (major and minor) pods and the assessment of their antioxidant activity. The V. faba methanolic extracts were characterized by HPLC-DAD-MS/MS for identification of polyphenolic compounds. The total phenolic content and antioxidant capacity of the extracts were evaluated by colorimetric methods (Folin-Ciocalteu, DPPH scavenging capacity assay, and FRAP assay). Main compounds identified by HPLC-DAD-MS/MS were derivatives of caffeic acid, coumaric acid and kaempferol. The broad bean Jögeva variety presented the highest content of free and esterified phenolics (26.3 and 26.7 mg 100 g-1 dry weight, respectively), followed by the horse bean varieties Bauska and Lielplatones. These results were corroborated by the analysis of total phenolic content, DPPH scavenging capacity and FRAP. This study confirmed the rich phenolic content of V. faba pods suggesting to be an interesting novel source for animal nutrition, promoting product quality and consumers' health.

Qualitative carbonyl profile in coffee beans through GDME-HPLC-DAD-MS/MS for coffee preliminary characterization.

In this work, an analytical methodology for volatile carbonyl compounds characterization in green and roasted coffee beans was developed. The methodology relied on a recent and simple sample preparation technique, gas diffusion microextraction for extraction of the samples' volatiles, followed HPLC-DAD-MS/MS analysis. The experimental conditions in terms of extraction temperature and extraction time were studied. A profile for carbonyl compounds was obtained for both arabica and robusta coffee species (green and roasted samples). Twenty-seven carbonyl compounds were identified and further discussed, in light of reported literature, with different coffee characteristics: coffee ageing, organoleptic impact, presence of defective beans, authenticity, human's health implication, post-harvest coffee processing and roasting. The applied methodology showed to be a powerful analytical tool to be used for coffee characterization as it measures marker compounds of different coffee characteristics.

Determination of ammonia nitrogen in solid and liquid high-complex matrices using one-step gas-diffusion microextraction and fluorimetric detection.

This paper introduces a new method for a one-step determination of ammonia nitrogen (NH3) in high complex solid and liquid samples from the agricultural and livestock sectors. To this end, we developed a simultaneous extraction and fluorimetric labeling of NH3, using gas diffusion microextraction (GDME), followed by the fluorescence measurement under 96-well microplate format. The GDME ensured a selective diffusion of NH3 through a commercial hydrophobic membrane, and confined the acceptor solution, which included the fluorimetric labeling reagent o-phthalaldehyde (OPA). The OPA-NH3 labeling reaction was optimized resorting to a full factorial experimental design, which showed that the reducing agent (Na2SO3) concentration was critical to achieve the highest sensitivity. A similar optimization approach for GDME showed that time and temperature significantly influenced the sensitivity of the assay, and also that the modifications in these two factors could be used to adjust the sensitivity according to the concentrations present in the samples. In our final conditions, it was possible to quantify NH3 in the range between 0.38 and 6.27mgL-1 using a 10min extraction at 25°C in different samples (e.g., corn and grass silages, feces, urine). The developed method showed a high repeatability and reproducibility (intraday and interday relative standard deviations values of 4.5% and 9.5%, respectively) and an adequate limit of detection (0.22mgL-1). This new methodology also highlighted the simplicity and versatility of GDME for the determination of volatile components of high-complex matrices, which will certainly drive future developments in the analysis of environmental and biological samples.

Gas-diffusion microextraction coupled with spectrophotometry for the determination of formaldehyde in cork agglomerates.

In this work, a simple methodology was developed for the extraction and determination of free formaldehyde content in cork agglomerate samples. For the first time, gas-diffusion microextraction was used for the extraction of volatile formaldehyde directly from samples, with simultaneous derivatization with acetylacetone (Hantzsch reaction). The absorbance of the coloured solution was read in a spectrophotometer at 412 nm. Different extraction parameters were studied and optimized (extraction temperature, sample mass, volume of acceptor solution, extraction time and concentration of derivatization reagent) by means of an asymmetric screening. The developed methodology proved to be a reliable tool for the determination of formaldehyde in cork agglomerates with the following suitable method features: low LOD (0.14 mg kg-1) and LOQ (0.47 mg kg-1), r 2 = 0.9994, and intraday and interday precision of 3.5 and 4.9%, respectively. The developed methodology was applied to the determination of formaldehyde in different cork agglomerate samples, and contents between 1.9 and 9.4 mg kg-1 were found. Furthermore, formaldehyde was also determined by the standard method EN 717-3 for comparison purposes; no significant differences between the results of both methods were observed. Graphical abstract Representation of the GDME system and its main components.

An Insight on Salting-out Assisted Liquid-Liquid Extraction for Phytoanalysis.

INTRODUCTION: Salting-out assisted liquid-liquid extraction (SALLE) is a technique that, although simple and not requiring any complex equipment, is very powerful and versatile. It has obtained growing interest in bioanalysis particularly when combined with chromatographic techniques. OBJECTIVES: Herein, fennel seeds (Foeniculum vulgare Mill.) were used as a case-study to show the application of SALLE in phytochemical analysis. MATERIAL AND METHODS: SALLE combined with HPLC-UV-MS/MS and GC-MS. RESULTS: By HPLC-UV-MS/MS analysis of the organic extract it was possible to identify various phenolic compounds, including quercetin derivatives, caffeic acid, p-coumaric acid and chlorogenic acid. The main compounds identified by GC-MS were estragole, fenchone, anisaldehyde, anethole, benzaldehyde, camphor and apiole. CONCLUSION: HPLC and GC analysis of the extracts showed that it is possible to isolate, in only one step, a wide range of compounds with distinct properties, allowing a detailed phytochemical analysis. Copyright © 2017 John Wiley & Sons, Ltd.

Isolation of Cells Specialized in Anticancer Alkaloid Metabolism by Fluorescence-Activated Cell Sorting.

Plant specialized metabolism often presents a complex cell-specific compartmentation essential to accomplish the biosynthesis of valuable plant natural products. Hence, the disclosure and potential manipulation of such pathways may depend on the capacity to isolate and characterize specific cell types. Catharanthus roseus is the source of several medicinal terpenoid indole alkaloids, including the low-level anticancer vinblastine and vincristine, for which the late biosynthetic steps occur in specialized mesophyll cells called idioblasts. Here, the optical, fluorescence, and alkaloid-accumulating properties of C. roseus leaf idioblasts are characterized, and a methodology for the isolation of idioblast protoplasts by fluorescence-activated cell sorting is established, taking advantage of the distinctive autofluorescence of these cells. This achievement represents a crucial step for the development of differential omic strategies leading to the identification of candidate genes putatively involved in the biosynthesis, pathway regulation, and transmembrane transport leading to the anticancer alkaloids from C. roseus.

Recent advances in salt-assisted LLE for analyzing biological samples.

Salt-assisted LLE (SALLE) has been attracting growing interest in bioanalysis. The technique is particularly advantageous due to its simple and fast experimental execution using conventional laboratory equipment. Besides, SALLE uses water-miscible organic solvents making the extracts readily compatible with various analytical separation and detection techniques. This article presents a brief overview of the extraction technique and its role in bioanalysis. Some of the most relevant achievements on SALLE application to biological samples are discussed - namely the study of the main extraction parameters, the combination with other extraction techniques and the instrumental analysis of the extracts. Developments on automation, miniaturization and microextraction for SALLE procedures are also discussed as a perspective for future applications even more attractive for the analysis of biological samples.