Recovery Of Metabolites Research Articles

Extraction Methods for Brain Biopsy NMR Metabolomics: Balancing Metabolite Stability and Protein Precipitation

Background/Objectives: Metabolic profiling of tissue samples via liquid-state nuclear magnetic resonance (NMR) requires the extraction of polar metabolites in a suitable deuterated solvent. Such methods often prioritise metabolite recovery over protein removal due to the relatively low sensitivity of NMR metabolomics and the routine use of methods able to supress residual protein signals. However, residual protein may impact metabolite integrity and the metabolite stability after NMR sample preparation is often overlooked. This study aimed to investigate the effect of residual protein contamination in rodent brain extracts and identify a reproducible extraction method that optimises metabolite recovery while ensuring sample stability. Methods: The performance of acetonitrile/water (50–100% MeCN), methanol/water (50–100% MeOH), and methanol/water/chloroform (MeOH/H2O/CHCl3) were assessed for extraction efficiency, reproducibility, residual protein contamination, and metabolite stability up to eight hours post NMR sample preparation. Results: Aspartate and glutamate deuteration were observed in 50% MeCN, 50% MeOH, and 67% MeOH extractions along with the conversion of N-acetyl aspartate to aspartate and acetate in 50% MeCN and 50% MeOH extractions. Both observations correlated with residual protein contamination and, thus, are a result of inadequate protein precipitation, as confirmed by ultrafiltration. MeOH/H2O/CHCl3 extraction preserved the stability of these metabolites while maintaining good extraction efficiency and reproducibility. Conclusions: Thus, we recommend MeOH/H2O/CHCl3 extraction for untargeted brain NMR metabolic profiling due to its effective protein precipitation and reliable performance. Nonetheless, the performance of detecting metabolites prone to oxidation such as ascorbate and glutathione is not improved by this method.

Urine organic acid metabolomic profiling by gas chromatography mass spectrometry: Assessment of solvent extract evaporation parameters on the recovery of key diagnostic metabolites

BackgroundAnalysis of urinary organic acids (UOAs) by gas chromatography mass-spectrometry (GC–MS) is widely used in metabolomic studies. It is a complex test with many limitations and pitfalls yet there is limited evidence in the literature to support best practice. This study investigated the impact of drying down time and temperature on the recovery of 16 key analytes from solvent extracts. MethodsPooled urine specimens were enriched with organic acids. Urine aliquots (n = 3) were acidified and extracted into diethyl ether and ethyl acetate. Extracts were dried under nitrogen at ambient temperature (25 °C); 40 °C; 60 °C then left for 0; +5; +15 min. Dried extracts were derivatised with N,O,-bis-(trimethylsilyl)trifluoroacetamide prior to analysis by GC–MS. Urine specimens from individuals with biotinidase deficiency, maple syrup urine disease (MSUD) and ketotic hypoglycemia were analysed to demonstrate the potential clinical impact. ResultsRecovery of shorter chain hydroxycarboxylic acids decreased significantly when extracts were dried above 25 °C (mean recovery 89 % at 60 °C, p < 0.01) or left under nitrogen post-drying (mean recovery at ambient + 15 min, 40 °C + 15mins and 60 °C + 15mins was 56 %, 12 % and 2 %, respectively, p < 0.01). Whilst dicarboxylic acids/medium chain fatty acids were unaffected by temperature (mean recovery 100 %), prolonged drying reduced recovery (mean recovery 85 % at 60 °C + 15mins, p < 0.01). ConclusionsEvaporation of solvent extracts with heat and/or prolonged drying under nitrogen results in significant losses of the shorter chain hydroxycarboxylic acids. The evaporation protocol must be carefully controlled to ensure accurate and reproducible results, preventing misdiagnoses and/or misinterpretation of results.

Recovery of metabolites via subnivean photosynthesis in Arctic tundra plants: Implications for climate change

AbstractPlants have evolved numerous strategies for surviving the harsh conditions of the Arctic. One strategy for Arctic evergreen and semi‐evergreen species is to photosynthesize beneath the snow during spring. However, the prevalence of this photosynthesis and how recent photosynthates are used is still unknown. Here we ask, how is newly acquired carbon beneath the snow allocated? To answer this question, we delivered isotopically labeled 13CO2 to tussock tundra plants before snowmelt. Soluble sugars and starches were preferentially enriched with 13C in all five species tested, with lipids having comparatively low 13C enrichment. These results provide evidence of the recovery of metabolites used over the long winter. Additionally, these new soluble sugars may function in photoprotection and cold tolerance as plants release from snow cover. Climate change, by reducing the duration of subnivean photosynthesis of these species, will limit metabolite production before snowmelt, which may lead to a reduction in the ability of these species to compete effectively during the growing season, potentially leading to changes in community structure.

Temperature-dependent exercise recovery is not associated with behavioral thermoregulation in a salmonid fish

The relationship between behavioral thermoregulation and physiological recovery following exhaustive exercise is not well understood. Behavioral thermoregulation could be beneficial for exercise recovery; for example, selection of cooler temperatures could reduce maintenance metabolic cost to preserve aerobic scope for recovery cost, or selection of warmer temperatures could accelerate recovery of exercise metabolites. While post-exercise behavioral thermoregulation has been observed in lizards and frogs, little is known about its importance in fish. We examined the influence of post-exercise recovery temperature on metabolic rate, thermal preference, and metabolite concentrations in juvenile brook char (Salvelinus fontinalis). Fish were acclimated to and exercised at 15 °C, then recovered at either 15 °C or 10 °C while their metabolic rate was measured via respirometry. Metabolite concentrations were measured in fish after exercise at 15 °C and recovery under one of three thermal treatments (to simulate various behavioral thermoregulation scenarios): (i) 6 h recovery at 15 °C, (ii) 6 h recovery at 10 °C, or (iii) 3 h recovery at 10 °C followed by 3 h recovery at 15 °C. Thermal preference was quantified using a static temperature preference system (15 °C vs. 10 °C). Metabolic rates returned to resting faster at 10 °C compared with 15 °C, although at 10 °C there was a tradeoff of delayed metabolite recovery. Specifically, post-exercise plasma osmolality, plasma lactate, and muscle lactate remained elevated for the entire period in fish recovering at 10 °C, whereas these parameters returned to resting levels by 6 h in fish from the other two recovery groups. Regardless, fish did not exhibit clear behavioral thermoregulation (i.e., fish overall did not consistently prefer one temperature) to prioritize either physiological recovery process. The advantage of metabolic rate recovery at cooler temperatures may balance against the advantage of metabolite recovery at warmer temperatures, lessening the usefulness of behavioral thermoregulation as a post-exercise recovery strategy in fish.

LC/TQ mass spectrometry analysis for estimation of residual behavior and dissipation kinetics of isocycloseram and its metabolite's in/on tomato (Solanum lycopersicum)

Isocycloseram is a new isoxazoline group pesticide with novel mode of action and very effective against various insect and mite species. The study was undertaken to determine the residual behavior of isocycloseram in/on tomato fruit and soil using liquid chromatography-tandem mass spectrometry. The limit of quantification and limit of detection were workout to be 0.01 and 0.003 μg/g, respectively for isocycloseram and the metabolites. The recovery of isocycloseram and metabolites were 94–105 % in tomato matrix and in soil matrix recovery was 93–101 % with < 20 % relative standard deviation. After applications of 60 and 75 g a.i./ha, the total initial residues of isocycloseram (including metabolites) on tomato fruits were 0.411 and 0.670 µg/g that dissipated to half in 3.13 and 3.46 days at respective doses. The hazard quotient computed for the insecticide was below one thus indicating its safety for humans.

Changes in rumen epithelial morphology and transcriptome, rumen metabolome, and blood biochemical parameters in lactating dairy cows with subacute rumen acidosis following rumen content transplantation

Interventions targeting the gut microbiota, such as fecal microbiota transplantation, prove effective in repairing the intestinal barrier and facilitating the recovery of its function and metabolism. However, the regulatory mechanisms governing the remodeling of rumen epithelial morphology and function, rumen metabolism, and host metabolism in cows of subacute ruminal acidosis (SARA) remain poorly understood. Here, we explored the changes in rumen epithelial morphology and transcriptome, rumen metabolome, and blood biochemical parameters in SARA cows following rumen content transplantation (RCT). The entire experiment consisted of 2 periods: the SARA induction period and the RCT period. During the SARA induction period, 12 ruminally cannulated lactating Holstein cows were randomly allocated into 2 groups, fed either a conventional diet [CON; n = 4; 40% concentrate, dry matter (DM) basis] or a high-grain diet (HG; n = 8; 60% concentrate, DM basis). Following the SARA induction period, the RCT period started. The HG cows were randomly assigned to 2 groups: the donor-recipient (DR) group and the self-recipient (SR) group. Rumen contents were entirely removed from both groups before RCT. For the DR group, cows were administered 70% rumen content from the CON cows, paired based on comparable body weight; for the SR group, each cow received 70% self-derived rumen content. The results revealed no significant differences in the thicknesses of the stratum corneum, granulosum, and spinosum/basale layers, as well as the total depth of the epithelium between the SR and DR groups. All these measurements exhibited a decreasing trend and fluctuations over time after the transfer. Notably, these fluctuations tended to stabilize at 13 or 16 d after RCT in the SR group, whereas they tended to stabilize after 8 or 13 d of transfer for the DR group. Transcriptome sequencing revealed that a total of 277 differentially expressed genes (DEGs) were identified between the 2 groups. Enrichment analysis showed that the DEGs were significantly enriched in 11 Gene Ontology biological processes and 14 KEGG pathways. The DEGs corresponding to almost any of these 11 biological process terms and 14 pathways showed mixed up- or downregulation following RCT. Metabolomics analysis indicated that a total of 33 differential metabolites were detected between the SR and DR groups, mainly enriched in 5 key metabolic pathways, including plant polysaccharides and starch degradation, lipid metabolism, amino sugar and nucleotide metabolism, purine metabolism, and Krebs cycle. Among them, the levels of differential metabolites associated with the degradation of plant polysaccharides and starches, metabolism of amino sugars and nucleotides, and purine metabolism pathways were significantly elevated in the DR cows. The results of blood biochemical parameters showed that the triglyceride concentration of the DR cows was increased than that of the SR cows, comparable to the level observed in the CON cows during the SARA induction period. Generally, our findings indicated that RCT facilitated the recovery of rumen epithelial morphological structure but did not promote its function recovery. Moreover, RCT enhanced rumen plant polysaccharide and starch degradation, amino sugar and nucleotide sugar metabolism, as well as purine metabolism. Additionally, it further promoted the recovery of plasma metabolites related to lipid metabolism.

Development of metabolome extraction strategy for metabolite profiling of skin tissue.

Changes in skin phenotypic characteristics are based on skin tissue. The study of the metabolic changes in skin tissue can help understand the causes of skin diseases and identify effective therapeutic interventions. We aimed to establish and optimize a non-targeted skin metabolome extraction system for skin tissue metabolomics with high metabolite coverage, recovery, and reproducibility using gas chromatography/mass spectrometry. The metabolites in skin tissues were extracted using eleven different extraction systems, which were designed using reagents with different polarities based on sequential solid-liquid extraction employing a two-step strategy and analyzed using gas chromatograph/mass spectrometry. The extraction efficiency of diverse solvents was evaluated by coefficient of variation (CV), multivariate analysis, metabolites coverage, and relative peak area analysis. We identified 119 metabolites and the metabolite profiles differed significantly between the eleven extraction systems. Metabolites with high abundances in the organic extraction systems, followed by aqueous extraction, were involved in the biosynthesis of unsaturated fatty acids, while metabolites with high abundances in the aqueous extraction systems, followed by organic extraction, were involved in amino sugar and nucleotide sugar metabolism, and glycerolipid metabolism. MeOH/chloroform-H2O and MeOH/H2O-chloroform were the extraction systems that yielded the highest number of metabolites, while MeOH/acetonitrile (ACN)-H2O and ACN/H2O-IPA exhibited superior metabolite recoveries. Our results demonstrated that our research facilitates the selection of an appropriate metabolite extraction approach based on the experimental purpose for the metabolomics study of skin tissue.

A novel biomonitoring method to detect pyrethroid metabolites in saliva of occupationally exposed workers as a tool for risk assessment

Saliva is valuable in exposure assessment having been successfully used for drug and environmental pollutant detection, providing a surrogate measure of plasma concentrations. Pyrethroid biomarkers have not previously been assessed in saliva, although are prime candidates for saliva detection. This study’s objectives were to (1) develop a liquid-liquid extraction (LLE) method to quantify six pyrethroid metabolites using gas chromatography/ion trap mass spectrometry and (2) assess its application for an occupationally exposed population. Several solvents and mixing protocols were optimized for metabolite recovery. The optimized method was applied to a population of pest control operators (PCOs) and compared against a urine sample before and after a full workday using pesticides. A questionnaire collected demographic information, occupational history, and occupational and non-occupational exposure data. LLE recoveries ranged from 85–104% and 72–88% for toluene and dichloromethane using slow mixing, and 49–103% for methyl tert-butyl ether by fast mixing. Urinary 3-Phenoxybenzoic acid (3PBA) was detected in 100% of pre- and post-work urine samples. Three PCOs had increased urinary pyrethroid metabolite levels post-work. Salivary 3PBA was present below detection limit in two of the three PCO’s post-work saliva samples, demonstrating that salivary 3PBA could be measured in PCOs after the workday. This study presents preliminary findings of a potential, low-risk biomonitoring technique that may be utilized in future occupational pyrethroid exposure and risk assessment research.

Understanding microbiome dynamics and functional responses during acidogenic fermentation of sucrose and sugarcane vinasse through metatranscriptomic analysis

Improving anaerobic digestion of sugarcane vinasse – a high-strength wastewater from ethanol distillation – is a subject of great interest, in view of the reduction of the pollutants and recovery of methane and valuable metabolites as byproducts. Through metatranscriptomic analysis, this study evaluated the active microbiome and metabolic pathways in a continuous acidogenic reactor: Stage 1S (control): 100% sucrose-based substrate (SBS); Stage 2SV (acclimation): 50% SBS and 50% vinasse; Stage 3V: 100% vinasse. Metatranscriptome obtained from each Stage was subjected to taxonomic and functional annotations. Under SBS feeding, pH dropped to pH 2.7 and biohydrogen production was observed. As vinasse was added, pH increased to 4.1–4.5, resulting in community structure and metabolite changes. In Stage 3V, biohydrogen production ceased, and propionate and acetate prevailed among the volatile fatty acids. Release of homoacetogenesis enzymes by Clostridium ljungdahlii and of uptake hydrogenase (EC 1.12.99.6) by Pectinatus frisingensis were linked to hydrogen consumption in Stages 2SV and 3V. Metabolic pathways of vinasse compounds, such as carbohydrates, malate, oxalate, glycerol, sulfate and phenol, were investigated in detail. In pyruvate metabolism, gene transcripts of oadA (oxaloacetate decarboxylase) and mdh (malate dehydrogenase), were upregulated in Stage 3V, being mostly attributed to P. frisingensis. Acetate formation from vinasse degradation was mainly attributed to Megasphaera and Clostridium, and propionate formation to P. frisingensis. Glycerol removal from vinasse exceeded 99%, and gene transcripts encoding for glpF (glycerol uptake facilitator protein), glpK (glycerol kinase) and glpABC (glycerol-3-phosphate dehydrogenase) were expressed mostly by Pectinatus and Prevotella. mRNA profiling showed that active bacteria and gene expression greatly changed when vinasse replaced sucrose, and Pectinatus was the main active bacterium degrading the searched compounds from vinasse. The identification of the main metabolic routes and the associated microorganisms achieved in this work contributes with valuable information to support further optimization of fermentation towards the desired metabolites.

Supercritical CO2 Extraction of Wine and Beer Yeast Residues for Sustainable Bioproduct Recovery

Abstract This paper presents a study on the use of supercritical carbon dioxide (SC-CO2) extraction to isolate yeast metabolites from beer and wine matrices effectively. A major contributor to the flavour and aroma profiles of alcoholic beverages, yeast contains a rich reservoir of bioactive compounds that can be used in various industries, including food and pharmaceuticals. Conventional extraction methods often fail to obtain comprehensive and high-quality recoveries of these valuable metabolites. In response, this study explores the use of SC-CO2, a green and tuneable solvent, to overcome the limitations of conventional extraction methods. The study investigates the influence of critical process parameters such as pressure, temperature, and extraction time on the yield and composition of extracted yeast metabolites. Advanced analytical methods (gas chromatography for the determination of fatty acids and high-performance liquid chromatography for the determination of the amino acid composition of the yeast residue left after extraction) are used in the analysis to characterize the chemical profile of the extracts. The results show that SC-CO2 extraction offers a promising alternative to extract a diverse range of yeast-derived compounds, including flavour-enhancing esters, phenolic compounds, and bioactive peptides. The optimized extraction conditions show a significant improvement in extraction efficiency compared to traditional methods. Sensory analysis reveals that beverages produced with SCCO2-extracted yeast have distinctive and desirable flavour profiles. This research not only contributes to the development of extraction technologies in the beverage industry but also opens up new opportunities for the use of yeast-derived bioactive compounds in various applications. The environmentally friendly nature of SC-CO2 extraction meets the growing demand for sustainable and clean technologies in food and beverage processing. Overall, the results of this study highlight the potential of SC-CO2 extraction as a valuable tool for improving the extraction of bioactive yeast metabolites, thereby influencing the quality and sensory properties of beer and wine.

Harvesting of Scenedesmus obliquus Cells by Chemical Flocculation: The Effects of Various Flocculants in Bio-recovery of Carotenoids

One of the major challenges in the downstream processing of microalgae is harvesting cells from the culture medium, which account for 20-30% of the production cost. Several harvesting methods and their combinations are available efficiency of which depends on algal sp. and biomass concentration. However, the bio recovery of algal metabolites for quite a number of harvesting methods is rarely reported. In the present study, an experiment was carried out to determine the effects of different flocculants (AlCl2, FeCl3 and ZnSO4, MgSO4) in harvesting Scenedesmus obliquus and its bio recovery of carotenoids. From the result obtained FeCl3 was the best flocculant harvesting up to 99.69% algal cells at the first 30 minutes of reaction time. Similarly, in terms of carotenoid bio-recovery, the highest carotenoids content was recorded in cells harvested with ferric chloride (i.e., 2.81 mg/ml) as compared to other coagulants.

Implementation of pure shift 1 H NMR in metabolic phenotyping for structural information recovery of biofluid metabolites with complex spin systems.

NMR spectroscopy is a mainstay of metabolic profiling approaches to investigation of physiological and pathological processes. The one-dimensional proton pulse sequences typically used in phenotyping large numbers of samples generate spectra that are rich in information but where metabolite identification is often compromised by peak overlap. Recently developed pure shift (PS) NMR spectroscopy, where all J-coupling multiplicities are removed from the spectra, has the potential to simplify the complex proton NMR spectra that arise from biosamples and hence to aid metabolite identification. Here we have evaluated two complementary approaches to spectral simplification: the HOBS (band-selective with real-time acquisition) and the PSYCHE (broadband with pseudo-2D interferogram acquisition) pulse sequences. We compare their relative sensitivities and robustness for deconvolving both urine and serum matrices. Both methods improve resolution of resonances ranging from doublets, triplets and quartets to more complex signals such as doublets of doublets and multiplets in highly overcrowded spectral regions. HOBS is the more sensitive method and takes less time to acquire in comparison with PSYCHE, but can introduce unavoidable artefacts from metabolites with strong couplings, whereas PSYCHE is more adaptable to these types of spin system, although at the expense of sensitivity. Both methods are robust and easy to implement. We also demonstrate that strong coupling artefacts contain latent connectivity information that can be used to enhance metabolite identification. Metabolite identification is a bottleneck in metabolic profiling studies. In the case of NMR, PS experiments can be included in metabolite identification workflows, providing additional capability for biomarker discovery.

Optimising a urinary extraction method for non-targeted GC–MS metabolomics

Urine is ideal for non-targeted metabolomics, providing valuable insights into normal and pathological cellular processes. Optimal extraction is critical since non-targeted metabolomics aims to analyse various compound classes. Here, we optimised a low-volume urine preparation procedure for non-targeted GC–MS. Five extraction methods (four organic acid [OA] extraction variations and a “direct analysis” [DA] approach) were assessed based on repeatability, metabolome coverage, and metabolite recovery. The DA method exhibited superior repeatability, and achieved the highest metabolome coverage, detecting 91 unique metabolites from multiple compound classes comparatively. Conversely, OA methods may not be suitable for all non-targeted metabolomics applications due to their bias toward a specific compound class. In accordance, the OA methods demonstrated limitations, with lower compound recovery and a higher percentage of undetected compounds. The DA method was further improved by incorporating an additional drying step between two-step derivatization but did not benefit from urease sample pre-treatment. Overall, this study establishes an improved low-volume urine preparation approach for future non-targeted urine metabolomics applications using GC–MS. Our findings contribute to advancing the field of metabolomics and enable efficient, comprehensive analysis of urinary metabolites, which could facilitate more accurate disease diagnosis or biomarker discovery.

In Search of Complementary Extraction Methods for Comprehensive Coverage of the Escherichia coli Metabolome.

Escherichia coli is an invaluable research tool for many fields of biology, in particular for the production of recombinant enzymes. However, the activity of many such recombinant enzymes cannot be determined using standard biochemical assays, as often, the relevant substrates are not known, or the products produced are not detectable. Today, the biochemical footprints of such unknown enzyme activities can be revealed via the analysis of the metabolomes of the recombinant E. coli clones in which they are expressed, using sensitive technologies such as mass spectrometry. However, before any metabolites can be identified, it is necessary to achieve as high a coverage of the potential metabolites present within E. coli as possible. We have therefore analyzed a wide range of different extraction methods against the cell free extracts of various recombinant E. coli clones. The results were analyzed to determine the minimum number of extractions that achieved high recovery and coverage of metabolites. Two methods were selected for further analysis due to their ability to produce not only high numbers of ions, but also wide mass coverage and a high degree of complementarity. One extraction method uses acetonitrile and water, in a 4:1 ratio, which is then dried down and reconstituted in the chromatography running buffer prior to injection onto the chromatography column, and the other extraction method uses a combination of methanol, water and chloroform, in a 3:1:1 ratio, which is injected directly onto the chromatography column. These two extraction methods were shown to be complementary to each other, as regards the respective metabolites extracted, and to cover a large range of metabolites.

A large-scale, targeted metabolomics method for the analysis and quantification of metabolites in human plasma via liquid chromatography-mass spectrometry

Metabolomics is the study of small molecules, primarily metabolites, that are produced during metabolic processes. Analysis of the composition of an organism's metabolome can yield useful information about an individual's health status at any given time. In recent years, the development of large-scale, targeted metabolomic methods has allowed for the analysis of biological samples using analytical techniques such as LC-MS/MS. This paper presents a large-scale metabolomics method for analysis of biological samples, with a focus on quantification of metabolites found in blood plasma. The method comprises a 10-min chromatographic separation using HILIC and RP stationary phases combined with positive and negative electrospray ionization in order to maximize metabolome coverage. Complete analysis of a single sample can be achieved in as little as 40 min using the two columns and dual modes of ionization. With 540 metabolites and the inclusion of over 200 analytical standards, this method is comprehensive and quantitatively robust when compared to current targeted metabolomics methods. This study uses a large-scale evaluation of metabolite recovery from plasma that enables absolute quantification of metabolites by correcting for analyte loss throughout processes such as extraction, handling, or storage. In addition, the method was applied to plasma collected from adjuvant breast cancer patients to confirm the suitability of the method to clinical samples.

The use of microalgae biomass for the production of marketable products. Part 4. The manufacture of biofuels from microalgae using bioengineering approaches

This is a review of the literature devoted to the production of microalgae biomass and its processing into marketable chemical products using advanced bioengineering approaches. The paper considers approaches to the manufacture of biofuels – biodiesel fuel, ethanol and hydrogen – from microalgae. Methods of biomass pretreatment, recovery of metabolites and their processing into biofuels are discussed.

Application of novel plasma separation filter cards for quantification of nucleoside/nucleotide reverse transcriptase inhibitor di/triphosphates in dried blood spots using LC-MS.

Background: A rapid and sensitive LC-MS method has been developed and validated for the quantification of nucleoside di/triphosphates using a novel plasma separation card (HemaSep). Materials & methods: Cards were spotted with whole blood and stored at -80°C. Metabolites were extracted using 70:30 MeOH:20% formic acid, followed by weak anion exchange SPE and eluted using a Biobasic-AX column. Quantification was performed using a triple quadrupole mass spectrometer with a calibration range of 1.25-250pmol/sample. Results: The recovery of metabolites was high (>93%). Precision and accuracy were acceptable and metabolites remained stable on the card after 29days (stored at ambient temperature). Conclusion: HemaSep dried blood spots are a useful microsampling tool and offer an alternative to liquid plasma as they maintain stability over time.

Comparative Study on Assisted Solvent Extraction Techniques for the Extraction of Biologically Active Compounds from Sideritis raeseri and Sideritis scardica.

The plants in the Sideritis genus are postulated to exhibit several important medicinal properties due to their unique chemical composition. To isolate the targeted phytochemical compounds, the selection of a suitable extraction method is of primary importance. In this work, a comparative study on the phytochemical profiles of various Sideritis raeseri and Sideritis scardica extracts has been carried out. An untargeted metabolomics approach based on ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry was applied to investigate the metabolic differences between extracts obtained by conventional extraction and extractions assisted by microwaves, ultrasounds and high pressure. Additionally, the influence of extraction solvents on HPLC antioxidant profiles obtained following the derivatization of analytes with ABTS reagent was evaluated. A total of 102 metabolites have been putatively identified. The major secondary metabolites groups were classified as flavonoids, terpenoids, phenylethanoid glycosides and phenolic acids. The main antioxidants in the extracts were isoscutellarein and hypolaetin derivatives as well as verbascoside and chlorogenic acid. The results showed that 70% ethanol was the most effective extractant for different classes of phytochemicals including antioxidants. In addition, extraction supported with microwaves, ultrasounds or high pressure improved the overall recovery of metabolites by about 3 times compared to the conventional extraction method.

A review on efforts for improvement in medicinally important chemical constituents inAconitum through biotechnological interventions.

The genus Aconitum belongs to the family Ranunculaceae, is endowed with more than 350 species on the earth. Medicinally important aconitine type of diterpenoid alkaloids are the characteristic compounds in most of the Aconitum species. The present review endeavored the major research carried out in the field of genetic resource characterization, pharmacological properties, phytochemistry, major factors influencing quantity, biosynthetic pathways and processing methods for recovery of active ingredients, variety improvement, propagation methods, and important metabolite production through cell/organ culture of various Aconitum species. More than 450 derivatives of aconitine-type C19 and C20-diterpenoid alkaloids along with a few other non-alkaloidal compounds, such as phenylpropanoids, flavonoids, terpenoids, and fatty acids, have been identified in the genus. A few Aconitum species and theircommon diterpenoid alkaloid compounds are also well characterized for analgesic, inflammatory and cytotoxic properties. However, the different isolated compound needs to be validated for supporting other traditional therapeutical uses of the plant species. Aconitine alkaloids shared common biosynthesis pathway, but their diversification mechanism remains unexplored in the genus. Furthermore, the process needs to be developed on secondary metabolite recovery, mass-scale propagation methods, and agro-technologies for maintaining the quality of products. Many species are losing their existence in nature due to over-exploitation or anthropogenic factors; thus, temporal monitoring of the population status in its habitat, and suitable management programs for ascertaining conservation needs to be developed.

Single-step extraction of bioactive compounds from cruciferous vegetable (kale) waste using natural deep eutectic solvents

Cruciferous vegetables such as kale contain various health-promoting phytochemicals, and leaves rejected from harvesting could be a valuable source of phytochemicals. Conventional organic solvents used for the recovery of these bioactive metabolites are hazardous, and therefore more benign equivalents are sought. This work explores the use of natural deep eutectic solvents (NADESs) with different hydrophilicity/hydrophobicity to recover polyphenols, carotenoids and chlorophylls from kale waste. Enhanced extraction of polyphenols was achieved by the aqueous solutions of hydrophilic glycerol-based NADESs, with yields of up to 2.2-fold higher than just using methanol. The antioxidant capacity of the aqueous extracts showed a strong correlation with their total phenolic contents. The best solvent in extracting polyphenols (glycerol:betaine at a molar ratio of 3:1) was further investigated to optimise its process conditions. The optimised extract provided the greatest stability of the bioactive polyphenols by retaining 91.7 and 88.6% of the original contents after 30 days of storage at 4 and 25 °C, respectively. Furthermore, this extraction approach was integrated with ethyl acetate, a bio-based solvent, to promote the simultaneous recovery of polyphenols (16.83 mg GAE (gallic acid equivalents) g−1 DW (dry weight)), carotenoids (0.91 mg g−1 DW) and chlorophylls (7.86 mg g−1 DW) from kale waste. After extraction, the immiscible aqueous NADES- and ethyl acetate-phases rich in polyphenols and carotenoids/chlorophylls, respectively, can be easily separated for different applications.