Extraction Of Metabolites Research Articles

A green approach for genistein and apigenin extraction optimization from by-products of soybean crops

Soybeans’ genistein and its isomer apigenin are widely studied bioactive compounds owing to their therapeutic potential for treating various health disfunctions. A green extraction of both from soy by-products could lead to a valuable and sustainable approach to adding value to these materials in a biorefinery context. Here Hansen Solubility Parameters (HSP) and the Conductor-like Screening MOdel for Real Solvents (COSMO-RS) were applied to screen green molecular solvents and Natural Eutectic Solvents for the extraction of genistein and apigenin from soy by-products. The predicted solubilities of genistein and apigenin in 18 shortlisted candidates were experimentally tested by dynamic maceration, the most industrially implemented natural products extraction technique. EtOH:H2O (8:2, v/v) and natural eutectic solvent (NAES) betaine:ethylene glycol (1:2, mol/mol) showed the highest performance. These were selected for extraction optimizations by Design of Experiments from soy branches, the largest by-product by mass. The optimum condition of each solvent was applied to extract all other parts of soy collected post-mechanical harvesting. The highest value of apigenin, 591.49 ± 26.7 μg/g, was achieved from soy pods with EtOH:H2O (8:2, v/v), while the highest of genistein, 54.04 ± 3.39 μg/g, was achieved from soybeans using the same solvent. Our findings highlight the necessity of exercising caution when interpreting in silico outcomes in the context of metabolite extractions from complex matrices. A trade-off between in silico solvent screening and experimental work should be followed when developing new phytochemical extraction processes. Furthermore, soy by-products emerged as competitive candidates for a long-term source of the bioactive apigenin in a biorefinery context.

Tracking the effect of roasting and fermentation on the metabolites of licorice root (Glycyrrhiza glabra L.) using UPLC-MS analysis combined with multivariate statistical analysis

BackgroundRoasting, honey-roasting and fermentation are the most common pre-processing procedures of licorice roots. They were shown to noticeably change the composition of extracts. In this work, the common alterations in licorice secondary metabolites by processing were interpreted. Comprehensive metabolic profiling of different studied samples was undergone.MethodsUPLC-QqQ-MS/MS analysis coupled to various chemometric analysis models was implemented to unravel the effect of different pre-processing procedures on the chemical profile of licorice samples.ResultsUPLC-QqQ-MS/MS analysis designated 133 chromatographic peaks with saponins, flavonoids, chalcones and pterocarpans being the most abundant groups. Triterpene saponins dominated the secondary metabolites in the aqueous extracts, with fermented samples showing the highest relative amounts. Meanwhile the ethanol extracts showed significant amounts of chalcones. Melanoidins were only detected in roasted and honey roasted samples. Multivariate models indicated that roasting of samples induced a greater effect on the polar metabolites rather than nonpolar ones. Variable of importance (VIP) plot indicated that glycyrrhizin and its hydrolysis product glycyrrhetinic acid, trihdroxychalcone diglycoside, glabrone and glabridin are the main chemical features responsible for the discrimination of samples.ConclusionCoupling UPLC-MS/MS to multivariate analysis was a successful tool that unveiled the significant effect of different pre-processing methods on the chemical profile of processed and unprocessed licorice samples. Moreover, such coupling unraveled the discriminatory chemical compounds among tested samples that can be employed as markers for the processing procedure of licorice.

Urinary metabolite signatures reflect the altered host metabolism in severe obstructive sleep apnea

Obstructive sleep apnea (OSA) is a common sleep-related breathing disorder. The onset and progression of OSA are often linked with severe cardiovascular and metabolic comorbidities. At the same time, given the increasing prevalence of OSA, novel methods to screen OSA and its follow-up are needed. Untargeted metabolic profiling of OSA patients and healthy controls was planned to capture a snapshot of urinary metabolites and potential biomarkers using the gas chromatography-mass spectrometry (GC–MS) method.Polysomnography (PSG) confirmed severe OSA patients with AHI index ≥ 30 were considered for urine sample collection. The sample size was constituted of OSA (n = 36) and healthy controls (n = 36). Metabolite extraction and derivatization were performed and metabolomic analysis was performed by using GC–MS.The obtained data set was statistically analyzed using univariate and multivariate analysis. The Orthogonal partial least-squares discriminant analysis (OPLS-DA) was performed to screen differential metabolites between OSA patients and healthy controls.The metabolomic analysis revealed a total of 142 significantly altered metabolites of interest.Biomarker analysis allows for the creation of a list of putative urinary biomarkers including GABA, malic acid, glutamic acid, epichoric acid etc., with an accuracy of 99.8 % to 100 % for OSA screening. Subsequently, pathway analysis revealed that related biochemical pathways like the tricarboxylic acid cycle (TCA), glutamate/glutamine, amino acid and fatty acid metabolism, that are significantly interlinked with these metabolic biomarkers can play a crucial role in the pathogenesis of OSA. This study paves the way to undertake mass screening in a larger population to identify specific and reliable biomarkers.

Application of Nanomaterials in the Production of Biomolecules in Microalgae: A Review.

Nanomaterials (NMs) are becoming more commonly used in microalgal biotechnology to empower the production of algal biomass and valuable metabolites, such as lipids, proteins, and exopolysaccharides. It provides an effective and promising supplement to the existing algal biotechnology. In this review, the potential for NMs to enhance microalgal growth by improving photosynthetic utilization efficiency and removing reactive oxygen species is first summarized. Then, their positive roles in accumulation, bioactivity modification, and extraction of valuable microalgal metabolites are presented. After the application of NMs in microalgae cultivation, the extracted metabolites, particularly exopolysaccharides, contain trace amounts of NM residues, and thus, the impact of these residues on the functional properties of the metabolites is also evaluated. Finally, the methods for removing NM residues from the extracted metabolites are summarized. This review provides insights into the application of nanotechnology for sustainable production of valuable metabolites in microalgae and will contribute useful information for ongoing and future practice.

Biomolecular condensates create phospholipid-enriched microenvironments.

Proteins and RNA can phase separate from the aqueous cellular environment to form subcellular compartments called condensates. This process results in a protein-RNA mixture that is chemically different from the surrounding aqueous phase. Here, we use mass spectrometry to characterize the metabolomes of condensates. To test this, we prepared mixtures of phase-separated proteins and extracts of cellular metabolites and identified metabolites enriched in the condensate phase. Among the most condensate-enriched metabolites were phospholipids, due primarily to the hydrophobicity of their fatty acyl moieties. We found that phospholipids can alter the number and size of phase-separated condensates and in some cases alter their morphology. Finally, we found that phospholipids partition into a diverse set of endogenous condensates as well as artificial condensates expressed in cells. Overall, these data show that many condensates are protein-RNA-lipid mixtures with chemical microenvironments that are ideally suited to facilitate phospholipid biology and signaling.

Emerging Technologies for the Discovery of Novel Diversity in Cyanobacteria and Algae and the Elucidation of Their Valuable Metabolites

Until recently, the study of cyanobacteria and microalgae has been hampered by the need to cultivate these organisms to gain insight into their cytomorphology, life cycle and molecular biology. However, various microbial species characterized by thick sheaths of exopolymeric substances were difficult to isolate in culture due to their associated symbiotic bacteria. Other microbes evaded culture. Such challenges have now been overcome by the development of metagenomic techniques that allow direct DNA sequencing from environmental samples, as well as high resolution microscopy techniques that permit direct imaging of environmental samples. The sampling of understudied taxa from extreme environments and of toxic species has been facilitated by specialized robotic equipment. Single-cell sequencing has allowed for the proper characterization of microalgal species and their response to environmental changes. Various strains of cyanobacteria, microalgae and macroalgae have gained renewed interest for their high-value metabolites. This paper provides an overview of the emerging technologies and explains how they are being used to identify such strains and their products for industrial application. Advances in genetic engineering and CRISPR technology have facilitated the production of strains that are more amenable to culture, metabolite extraction, scale-up and application in biorefinery approaches. Emerging analytical techniques are discussed, with the advent of multiomics and its application in this field.

Exploration of the aptitude to alleviate oxidative impairment and curb colorectal cancer manifestation by Nostoc calcicola in HT-29 adenocarcinoma cells

BackgroundMarine cyanobacteria have been known to contain several unique bioactive compounds which have different therapeutic potentials. The current research focuses to identify the efficacy of Nostoc calcicola to counteract the harmful effects of free radicals and testing its anticancer activity against colorectal adenocarcinoma cells (HT-29).ResultsMethanol is used as a solvent for the extraction of bioactive metabolites from Nostoc calcicola followed by phytochemical screening representing the presence of flavonoids, phenols, tannins, saponins, and steroids to find out bioactive metabolites. Furthermore, evaluation of the extract efficacy revealed the profound ability of Nostoc calcicola to scavenge free radicals by neutralizing different reactive oxygen species. At 100 µg/mL concentration, it inhibited DPPH radicals (73.4%), enhanced phosphomolybdenum reduction (53.5%), displayed ferric-reducing power (55.1%), and finally the extract revealed remarkable hydroxyl radicals scavenging capacity (94.8%), compared to the standards. These compelling results emphasize the robust antioxidant potential of the Nostoc calcicola extract. In vitro, studies demonstrated the selective cytotoxic effects of methanol extracts of Nostoc calcicola on the HT-29 human colorectal cancer cell line, as indicated by IC50 values of 25 µg/mL for the extracts. Treatment with me Nostoc calcicola decreased the cell viability of HT-29 cells followed by consistent morphological changes leading to cytotoxicity. Nuclear condensation and DNA fragmentation were observed using AO/EtBr and DAPI staining. Flow cytometry analysis further confirmed the incidence of apoptosis during the S phase of the cell cycle. Furthermore, western blotting analysis confirmed the activation of caspase 9, a pivotal enzyme in the intrinsic apoptosis pathway, suggesting the ability of Nostoc calcicola to induce apoptosis in HT-29 colorectal cancer cells.ConclusionThese findings underscore the potential of Nostoc calcicola as a valuable source of bioactive compounds with antioxidant and anticancer properties, warranting further investigation for their potential therapeutic applications in colorectal cancer.Graphical

Comparative Evaluation of Antioxidant and Anti-Depressant Activity of Macrotyloma Uniflorum vs. Clitoria Ternatea Methanolic Leaf Extracts

Background: Depression and anxiety are prevalent psychiatric disorders in the modern era, often attributed to the alterations in signalling neurotransmitters or the release of biogenic amines characterized by changes in consciousness, psychomotor functions, and emotional disturbances. Purpose: Several antidepressant drugs are available; they may cause irreversible adverse effects. Therefore, exploring herbal remedies as an alternative therapy holds potential benefits. Macrotyloma uniflorum and Clitoria ternatea are among the plants traditionally utilized for their anti-depression activity. Method: This study aims to compare the antidepressant activity of the methanolic extracts of M. uniflorum and C. ternatea leaves. Their phytochemical compositions were analysed, and antioxidant activities were evaluated using DPPH and Superoxide radical scavenging assays. Results: The outcomes of phytochemical screening indicated the presence of secondary metabolites in both extracts, including alkaloids, saponins, glycosides, steroids, flavonoids, phenols, terpenoids and tannins. The antioxidant assays indicated significant antioxidant potential in both M. uniflorum and C. ternatea extracts. Various biochemical assays, behavioural studies, and histopathological evaluations were conducted using a toluene-induced mice model. Conclusion: The behavioural study and biochemical estimations confirmed the antidepressant potential of both extracts. However, the M. uniflorum extract exhibited better efficacy in alleviating depressive symptoms. In light of future perspectives, it would be valuable to isolate and identify the specific compounds facilitating a more targeted pharmacological approach for depression pharmacotherapy.

Identify production area, growth mode, species, and grade of Astragali Radix using metabolomics “big data” and machine learning

BackgroundAstragali Radix (AR) is a widely used herbal medicine. The quality of AR is influenced by several key factors, including the production area, growth mode, species, and grade. However, the markers currently used to distinguish these factors primarily focus on secondary metabolites, and their validation on large-scale samples is lacking. PurposeThis study aims to discover reliable markers and develop classification models for identifying the production area, growth mode, species, and grade of AR. MethodsA total of 366 batches of AR crude slices were collected from six provinces in China and divided into learning (n = 191) and validation (n = 175) sets. Three ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) methods were developed and validated for determining 22 primary and 10 secondary metabolites in AR methanol extract. Based on the quantification data, seven machine learning algorithms, such as Nearest Neighbors and Gradient Boosted Trees, were applied to screen the potential markers and build the classification models for identifying the four factors associated with AR quality. ResultsOur analysis revealed that secondary metabolites (e.g., astragaloside IV, calycosin-7-O-β-D-glucoside, and ononin) played a crucial role in evaluating AR quality, particularly in identifying the production area and species. Additionally, fatty acids (e.g., behenic acid and lignoceric acid) were vital in determining the growth mode of AR, while amino acids (e.g., alanine and phenylalanine) were helpful in distinguishing different grades. With both primary and secondary metabolites, the Nearest Neighbors algorithm-based model was constructed for identifying each factor of AR, achieving good classification accuracy (>70%) on the validation set. Furthermore, a panel of four metabolites including ononin, astragaloside II, pentadecanoic acid, and alanine, allowed for simultaneous identification of all four factors of AR, offering an accuracy of 86.9%. ConclusionOur findings highlight the potential of integrating large-scale targeted metabolomics and machine learning approaches to accurately identify the quality-associated factors of AR. This study opens up possibilities for enhancing the evaluation of other herbal medicines through similar methodologies, and further exploration in this area is warranted.

Antimicrobial Efficacy of Allium cepa and Zingiber officinale Against the Milk-Borne Pathogen Listeria monocytogenes.

Listeria monocytogenes is an important food-borne pathogen that causes listeriosis and has a high case fatality rate despite its low incidence. Medicinal plants and their secondary metabolites have been identified as potential antibacterial substances, serving as replacements for synthetic chemical compounds. The present studies emphasize two significant medicinal plants, Allium cepa and Zingiber officinale, and their efficacy against L. monocytogenes. Firstly, a bacterial isolate was obtained from milk and identified through morphology and biochemical reactions. The species of the isolate were further confirmed through 16S rRNA analysis. Furthermore, polar solvents such as methanol and ethanol were used for the extraction of secondary metabolites from A. cepa and Z. officinale. Crude phytochemical components were identified using phytochemical tests, FTIR, and GC-MS. Moreover, the antibacterial activity of the crude extract and its various concentrations were tested against L. monocytogenes. Among all, A. cepa in methanolic extracts showed significant inhibitory activity. Since, the A. cepa for methanolic crude extract was used to perform autography to assess its bactericidal activity. Subsequently, molecular docking was performed to determine the specific compound inhibition. The docking results revealed that four compounds displayed strong binding affinity with the virulence factor Listeriolysin-O of L. monocytogenes. Based on the above results, it can be concluded that the medicinal plant A. cepa has potential antibacterial effects against L. monocytogenes, particularly targeting its virulence.

Secondary metabolites of mangrove-derived endophytic fungus, Pseudopestalotiopsis species investigated for antimicrobial and antioxidant activities

Attention is being diverted toward the bioprospecting of newer bioactive compounds from marine endophytic fungi. This is because marine fungi have shown a large chemo-diversity of untapped important secondary metabolites needed for drug development. In the present study, the secondary metabolites of a mangrove-derived endophytic fungus Pseudopestalotiopsis species isolated from the root of Rhizophora racemosa were investigated for antimicrobial and antioxidant activities. The fungal isolation, taxonomic identification, fermentation, extraction, and isolation of the fungal secondary metabolites were carried out using standard techniques. The fermentation product was subjected to fractionation. The crude extract and its fractions were screened for antimicrobial and antioxidant assays. The active extracts and fractions exhibited good antimicrobial activities against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas oleaginous, and Candida albicans with MIC values that ranged between 0.06 to 1 mg/mL. The Gram negatives were the most susceptible bacteria while C. albicans was the most susceptible fungi. Moderately low antioxidant activities were recorded in the DPPH and FRAP assays. The chromatographic separation and HPLC analysis of the fungal secondary metabolites yielded compounds: Palitantin (A), Cytosporin D (B), Cytosporin K (C), and Fusarielin (D). These compounds have been previously reported to possess varying pharmacological activities which include antimicrobial and antioxidant activities. Thus, The results of this study show that these compounds may, in part, account for the anti-microbial and antioxidant effect of the root of Rhizophora racemosa ethno medically.

Quantitative Analysis of Phytoconstituents of Hydroalcoholic Extract of Carica Papaya Leaf

Carica papaya (C. papaya) leaves have been used in folk medicine for centuries. Several reports are available for use of Carica papaya leaves in increasing blood platelet count in dengue hemorrhagic fever. Recent studies also reported that Carica papaya leaves exhibits anti-tumor activity and immunomodulatory effects. Papaya leaf extract is reported to contain carbohydrates, amino acids, sterols, saponin glycosides, iridoids, flavonoids, phenolics, anthraquionones, triterpenes and alkaloids. The present investigation demonstrates the concentration of secondary metabolites in the hydroalcoholic extract of Carica papaya leaves. The total phenolics, total alkaloid and total flavonoid content were calculated by Folin–Ciocalteu colorimetric method, Bromocresol green colorimetric method and aluminum chloride colorimetric method respectively.

In vitro antimicrobial activity of the crude extract of the endophytic bacterium Pseudomonas aeruginosa (SS93) isolated from Sapindus saponaria L.

Endophytes colonize the interior of plant tissues without causing any damage to their hosts. The plant Sapindus saponaria L., popularly known as ‘sabão-de-soldado’, presents a diversified endophytic microbiota and also medicinal properties. Endophytic microorganisms may produce secondary metabolites with different biotechnological properties. The present study aimed to evaluate the in vitro antibacterial and antifungal capacity of the crude extract of secondary metabolites produced by the endophytic bacteria P. aeruginosa SS93 isolated from S. saponaria leaves. The metabolites extract was obtained using the organic solvent ethyl acetate, and the antimicrobial activities were tested against six pathogenic bacteria (Enterococcus faecalis [ATCC 29212], Pseudomonas aeruginosa [ATCC 27853], Shigella flexneri [ATCC 12022], Salmonella enterica [CCCD a016], Escherichia coli [ATCC 25922], and Staphylococcus aureus [ATCC 25923]), and pathogenic fungi (Fusarium oxysporum, Glomerella sp., Sphaceloma sp., Fusarium solani, Maniliophtora perniciosa, and Sclerotinia sclerotiorum), by agar diffusion method. In the antibacterial assay, the best results were obtained against E. faecalis and S. aureus, where the formation of inhibition halos was observed in all tested concentrations, especially at 500 and 700 µg mL-1. Positive inhibitory activity against phytopathogenic fungi was observed, with the highest inhibition recorded against F. oxysporum (61.1%), followed by Sphaceloma sp. (55.7%), M. perniciosa (35.6%), F. solani (34.4%), and Glomerella sp. (30.4%).

Phytotoxic fungal secondary metabolites as herbicides.

Among the alternatives to synthetic plant protection products, biocontrol appears as a promising method. This review reports on the diversity of fungal secondary metabolites phytotoxic to weeds and on the approach generally used to extract, characterize, identify and exploit them for weed management. The 183 phytotoxic fungal secondary metabolites discussed in this review fall into five main classes of molecules: 61 polyketides, 53 terpenoids, 36 nitrogenous metabolites, 18 phenols and phenolic acids, and 15 miscellaneous. They are mainly produced by the genera Drechslera, Fusarium and Alternaria. The phytotoxic effects, more often described by the symptoms they produce on plants than by their mode of action, range from inhibition of germination to inhibition of root and vegetative growth, including tissue and organ alterations. The biochemical characterization of fungal secondary metabolites requires expertise and tools to carry out fungal cultivation and metabolite extraction, phytotoxicity tests, purification and fractionation of the extracts, and chemical identification procedures. Phytotoxicity tests are mainly carried out under controlled laboratory conditions (not always on whole plants), while effectiveness against targeted weeds and environmental impacts must be assessed in greenhouses and open fields. These steps are necessary for the formulation of effective, environment-friendly fungal secondary metabolites-derived bioherbicides using new technologies such as nanomaterials. This article is protected by copyright. All rights reserved.

Antibacterial properties and characterization of metabolite extracts of mangrove soil-associated bacteria from a subtropical island: Evaluation of their activity against common human pathogens and biofilms of clinical importance

We are now in the "post-antibiotic era" where there is a crucial need to develop new antibiotics with novel modes of action. Mangrove ecosystems are unique and shelter diverse halophilic microorganisms that can potentially produce novel antibacterial metabolites. In this work, extracts from four mangrove-derived isolates, namely Z3RZ1, Z3RZ3, Z3PZ1 and Z5PZ2, were tested for potential antibacterial activity against six pathogens: Escherichia coli (ATCC 25922), P. aeruginosa (ATCC 27853), Bacillus cereus (ATCC 11778), Staphylococcus aureus (ATCC 29213), Staphylococcus epidermidis (ATCC 12228) and Klebsiella pneumonia (ATCC 13883). The four extracts were tested for their antibiofilm potential against E. coli (ATCC 25922) and P. aeruginosa (ATCC 27853). The extract with the best antibiofilm activity was chemically characterized by liquid chromatography-mass spectrometry (LC-MS). Sequence analyses of the four isolates showed closest similarity matches to Micrococcus luteus (Z3RZ1), Globicatella sanguinis (Z3RZ3), Shewanella indica (Z3PZ1), and Shewanella indica (Z5PZ2). The Z3RZ3 extract had the strongest antibacterial activity against five pathogens, with the lowest minimum inhibitory concentrations (MIC) ranging from 3.91 × 102 to 6.250 × 103 µg/ml. All four extracts showed strong activity (>75%) and very strong activity (>95%) against E. coli and a P. aeruginosa biofilm formation respectively. Z3RZ3 extract was by far the most potent in disrupting pre-formed P. aeruginosa biofilms, displaying 100% efficacy within a 2-hr period. The Z3RZ3 extract contained cyclopentenones, terpenes, anthraquinone, thioinosine, cyclic lactam, benzene derivatives, and macrolides. Our study highlights the potential of the mangrove environment as a mine of new bioactive compounds with antibacterial and antibiofilm activities.

Ethno-botanical Verification and Phytochemical Profile of Ethanolic leaves Extract of Two Medicinal Plants (Phragmenthera capitata and Lantana camara) used in Nigeria using GC- MS Technique

Background: The present experiment was designed to determine the bioactive constituents from extracts of leaves of Phragmenthera capitata and Latana camara. The medicinal value of a plant species is dependent upon its various phytochemical constituents. Methodology:The chemical compositions of the ethanolic extract of the leaves of P. capitata and L. camara were investigated using theGas Chromatography-Mass spectrometry model GCMS-QP2010 PLUS SHIMADZU. The column used was aPerkin Elmer Elite – 5 capillary columns measuring 30m × 0.25mm with a film thickness of 0.25mm composed of 95% Dimethylpolysiloxane. The carrier gas used was Helium at a flow rate of 0.5ml / min. Results:Fifteen (15) bioactive phytochemical compounds were identified for P. capitata and twenty (20) bioactive phytochemical compounds were identified for L. camara. The prevailing compounds in P. capitata were, 3-H-pyrazol-3-1, Decanoic acid, n-Hexadecanoic acid, Pentanoic acid, Methyloctanoic acid, 4-octanoicacid, 15 -tetraosenoic acid, oleic acid, Octadecanoic acid, Pentafluoropropronic acid, (E)-13-docosenoic acid, Vitamin E, Lupeol and Chilosyphone. Also, in L. camara the following compounds were identified, 3-n-hexythiolane, Docosanoic acid, 1- nonanol, 1-hexacosanol, Divalonic acid, Decanoic acid, Octanoic acid, Eicosanoic acid, Hexadecanoic acid, Nonadecanoic acid, 9-12 octadecanoic acid, 11 -octadecadienoic acid, Pentadecanoic acid, Oleic acid, Methyl -Beta-D-arabinopyranoside, Acetic acid, Pentanoic acid, Heneicosanoic acid, methyl ester and 2H-pyran,2-(7-heptadecynloxytetrahydro. Conclusion: Based on the results of findings, it could be concluded that extract from P. capitata and L. camara may have antioxidant, antimicrobial, anti-cancer, anti-diabetic, anti-fungal and anti-convulsant activities due to the presence of secondary metabolites in the ethanolic extract

Endo metabolomic profiling of flor and wine yeasts reveals a positive correlation between intracellular metabolite load and the specific glycolytic flux during wine fermentation.

This study explored the intracellular metabolic variations between 17 strains of Saccharomyces cerevisiae belonging to two different genetic populations: flor and wine yeasts, in the context of alcoholic fermentation. These two populations are closely related as they share the same ecological niche but display distinct genetic characteristics. A protocol was developed for intracellular metabolites extraction and 1H-NMR analysis. This methodology allowed us to identify and quantify 21 intracellular metabolites at two different fermentation steps: the exponential and stationary phases. This work provided evidence of significant differences in the abundance of intracellular metabolites, which are strain- and time-dependent, thus revealing complex interactions. Moreover, the differences in abundance appeared to be correlated with life-history traits such as average cell size and specific glycolytic flux, which revealed unsuspected phenotypic correlations between metabolite load and fermentation activity.

Human Cerebrospinal Fluid Sample Preparation and Annotation for Integrated Lipidomics and Metabolomics Profiling Studies

Cerebrospinal fluid (CSF) is a metabolically diverse biofluid and a key specimen for exploring biochemical changes in neurodegenerative diseases. Detecting lipid species in CSF using mass spectrometry (MS)-based techniques remains challenging because lipids are highly complex in structure, and their concentrations span over a broad dynamic range. This work aimed to develop a robust lipidomics and metabolomics method based on commonly used two-phase extraction systems from human CSF samples. Prioritizing lipid detection, biphasic extraction methods, Folch, Bligh and Dyer (B&D), Matyash, and acidified Folch and B&D (aFolch and aB&D) were compared using 150 μL of human CSF samples for the simultaneous extraction of lipids and metabolites with a wide range of polarity. Multiple chromatographical separation approaches, including reversed-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC), and gas chromatography (GC), were utilized to characterize human CSF metabolome. The aB&D method was found as the most reproducible technique (RSD < 15%) for lipid extraction. The aB&D and B&D yielded the highest peak intensities for targeted lipid internal standards and displayed superior extracting power for major endogenous lipid classes. A total of 674 unique metabolites with a wide polarity range were annotated in CSF using, combining RPLC-MS/MS lipidomics (n = 219), HILIC-MS/MS (n = 304), and GC-quadrupole time of flight (QTOF) MS (n = 151). Overall, our findings show that the aB&D extraction method provided suitable lipid coverage, reproducibility, and extraction efficiency for global lipidomics profiling of human CSF samples. In combination with RPLC-MS/MS lipidomics, complementary screening approaches enabled a comprehensive metabolite signature that can be employed in an array of clinical studies.Graphical abstract

Toxicological effects of aqueous extract of Genipa americana L. leaves on adult zebrafish (Danio rerio): Chemical profile, histopathological effects and lack of genotoxicity

Genipa americana is a native plant of Brazil with potential applications in folk medicine. Whereas most of the phytochemical and pharmacological studies on this plant have focused on its fruits, the crude extracts of its leaves contain chemical metabolites that may have toxicity to organisms, which have yet to be investigated. This study aimed to determine the main groups of secondary metabolites in the aqueous extract of the leaves of G. americana by phytochemistry and qualitative HPLC, and to evaluate the possible toxicological effects and histopathological changes caused by this extract in zebrafish (Danio rerio) adults, through micronucleus test, nuclear abnormalities and histopathological analyses of gills and liver. While three metabolites of high intensity (phenolic compounds, flavonoids and triterpenes) were found in the phytochemical evaluation, the HPLC showed results compatible with flavonoids and iridoids, all belonging to common classes for this species and the Rubiaceae family. The acute toxicity test did not induce mortality or genotoxicity in zebrafish, but after exposure for 96 h, it was possible to observe injuries to the fish gill tissue, such as lamellar fusion, vasodilation and telangiectasia; in the liver, necrosis was visualized at 40 mg/L, and at higher concentrations (80 and 100 mg/L) induced sinusoidal widening was identified. In conclusion, the results demonstrated the toxic potential of this plant for aquatic species.

Gas Chromatography-Mass Spectrometry-Based Targeted Metabolomics of Hard Coral Samples.

Gas chromatography-mass spectrometry (GC-MS)-based approaches have proven to be powerful for elucidating the metabolic basis of the cnidarian-dinoflagellate symbiosis and how coral responds to stress (i.e., during temperature-induced bleaching). Steady-state metabolite profiling of the coral holobiont, which comprises the cnidarian host and its associated microbes (Symbiodiniaceae and other protists, bacteria, archaea, fungi, and viruses), has been successfully applied under ambient and stress conditions to characterize the holistic metabolic status of the coral. However, to answer questions surrounding the symbiotic interactions, it is necessary to analyze the metabolite profiles of the coral host and its algal symbionts independently, which can only be achieved by physical separation and isolation of the tissues, followed by independent extraction and analysis. While the application of metabolomics is relatively new to the coral field, the sustained efforts of research groups have resulted in the development of robust methods for analyzing metabolites in corals, including the separation of the coral host tissue and algal symbionts. This paper presents a step-by-step guide for holobiont separation and the extraction of metabolites for GC-MS analysis, including key optimization steps for consideration. We demonstrate how, once analyzed independently, the combined metabolite profile of the two fractions (coral and Symbiodiniaceae) is similar to the profile of the whole (holobiont), but by separating the tissues, we can also obtain key information about the metabolism of and interactions between the two partners that cannot be obtained from the whole alone.