GC-MS Assay Research Articles

Chemical Characterization and In Vitro Evaluation of Glucans from Fermentation-Produced Nutraceutical Bionutri-AR1®: Antioxidant and Immunomodulatory Properties

Background: The consumption of nutraceuticals or food supplements has increased crucially, aiming to address nutrient deficits and enhance immune system function. To develop safe food products with unique nutritional and functional benefits, new production methods of these nutraceuticals such as the fermentative process have been gaining prominence for industrial applications. Bionutri-AR1® is a nutraceutical produced via this bioprocess, featuring a complex composition, that has been used to improve the immune systems of debilitated people. Objectives: Considering the various biological properties attributed to glucans, one of its main components, this study aims to structurally characterize and evaluate, in vitro, the antioxidant and immunomodulatory potential of the polymers from this nutraceutical to assess whether these polymers contribute to the product’s reported biological effects. Methods/Results: Unlike previous reports, this study characterized by NMR, GC-MS, and Congo Red assay techniques two main glucans: a water-insoluble linear α-D-glucan with glycosidic bonds (1→4) and a soluble branched (1→3)- and (1→6)-linked β-glucan with a triple helix. Both glucans showed significant antioxidant activity, measured by their capacity to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. They were also capable of inducing the secretion of cytokines such as tumoral necrosis factor-alpha (TNF-α), interleukin 10 (IL-10), and interleukin 6 (IL-6), determined through capture enzyme-linked immunosorbent assay (ELISA), especially when co-stimulated with lipopolysaccharide (LPS). Conclusions: This suggests a dual action of these glucans in both proinflammatory and regulatory pathways. Future studies will describe the mechanisms by which these glucans, especially the insoluble ones, enhance immune system function, highlighting their potential use in immunotherapy.

Phytochemical Characterization, Antioxidant, and Antimicrobial Activity of the Vegetative Buds from Romanian Spruce, Picea abies (L.) H. Karst.

This study aims to investigate the vegetative buds from Picea abies (spruce), naturally found in a central region of Romania, through a comprehensive analysis of the chemical composition to identify bioactive compounds responsible for pharmacological properties. Using HPLC/derivatization technique of GC-MS and quantitative spectrophotometric assays, the phenolic profile, and main components of an ethanolic extract from the buds were investigated. The essential oil was characterized by GC-MS. Moreover, the antioxidant activity with the DPPH method, and the antimicrobial activity were tested. Heavy metal detection was performed by graphite furnace atomic absorption spectrometry. The main components of the alcoholic extract were astragalin, quercetin, kaempferol, shikimic acid, and quinic acid. A total content of 25.32 ± 2.65 mg gallic acid equivalent per gram of dry plant (mg GAE/g DW) and of 10.54 ± 0.083 mg rutin equivalents/g of dry plant (mg RE/g DW) were found. The essential oil had D-limonene, α-cadinol, δ-cadinene, 13-epimanool, and δ-3-carene as predominant components. The spruce vegetative buds exhibited significant antioxidant activity (IC50 of 53 μg/mL) and antimicrobial effects against Staphylococcus aureus. Furthermore, concentrations of heavy metals Pb and Cd were below detection limits, suggesting that the material was free from potentially harmful contaminants. The results confirmed the potential of this indigenous species to be used as a source of compounds with pharmacological utilities.

Harnessing Bacillus cereus from Surabaya Seawater for Enhanced Diesel Fuel Bioremediation in Tropical Ocean

Hydrocarbon is one of the primary organic pollutants that contaminate seawater. Most hydrocarbons which pollute the sea waters of Indonesia are the constituents of diesel fuel. This condition emphasizes the need to develop a bioremediation strategy for diesel fuel pollution in the country. A successful plan for bioremediation is primarily determined by the choice of the bacterial agent, which can be isolated from the polluted seawater. Furthermore, the setup must consider the characteristics and capability of the bacterial isolates to degrade diesel fuel. This study thus aimed to analyze the potential of Bacillus cereus isolated from the polluted seawater of Surabaya, East Java, Indonesia, as a bioremediation agent. The results of bacterial density, pH measurements, TPH and GC-MS assays indicated that the locally isolated Bacillus cereus could significantly reduce the hydrocarbon concentrations. More importantly, after 14 days of bioremediation, the polluting diesel fuel was categorized as biodegradable based on the BOD:COD ratio. This study shows that Bacillus cereus has excellent potential for bioremediating hydrocarbon contaminants in tropical seawater. HIGHLIGHTS Bacillus cereus from Surabaya's polluted waters fights diesel pollutants effectively. Bacillus cereus degrades hydrocarbons efficiently over two weeks, proving its bioremediation potential. Rise in BOD:COD ratio indicates Bacillus cereus' effective reduction of complex organics. GC-MS shows Bacillus cereus transforms hydrocarbons into shorter chains systematically. Bacillus cereus offers an eco-friendly solution for diesel pollution in tropical climates, urging further exploration. GRAPHICAL ABSTRACT

Chemical Composition and Antibacterial and Antiulcerative Colitis Activities of Essential Oil from Pruni Semen.

This study was sought to investigate the chemical composition and antibacterial and antiulcerative colitis (UC) effects of essential oil from Pruni Semen (PSEO). A GC-MS assay showed that the major compounds in PSEO were products of amygdalin hydrolysis, which possessed great antibacterial and anti-inflammatory potential. In vitro antibacterial experiments demonstrated that PSEO treatment inhibited activity of four kinds of intestinal pathogens probably by disrupting the cell wall. Further in vivo studies showed that PSEO administration significantly improved physiological indexes, attenuated histopathological characteristics, and inhibited proinflammatory cytokine production in dextran sulfate sodium (DSS)-induced UC mice. Network pharmacology and molecular docking results predicted that PSEO might prevent UC via regulating the PI3K/AKT pathway. Western blotting and immunofluorescence assays were further conducted for verification, and the results evidenced that PSEO intervention significantly regulated the PI3K/AKT pathway and the expression of its downstream proteins in DSS-induced mice. PSEO might provide a new dietary strategy for UC treatment.

Investigating serum concentration profiles of orally ingested short-chain fatty acid supplements.

Acetate, propionate, and butyrate are naturally-occurring short-chain fatty acids (SCFAs) derived from bacterial metabolism of dietary fibre and have been associated with numerous positive health outcomes. All three acids have been shown to offer unique physiological and metabolic effects and, therefore, could be targeted for co-ingestion as part of a nutritional/medicinal plan. However, a better understanding of the outcomes of supplementing in combination on circulating concentration profiles is necessary to confirm uptake efficacy. This study sought to investigate the acute circulating concentration profiles of acetate, propionate, and butyrate following oral supplementation. Three experimental trials were conducted including investigations to understand the impact of capsule coating on circulating concentration profiles, the effect of supplementation dose on uptake kinetics, and the outcome of a short, repeated, supplementation routine on circulating levels. Serum samples were analysed for SCFA content using a quantitative GC-MS assay. It was observed that an acid-resistant coated capsule caused a delayed and blunted blood concentration response, with the non-acid resistant trial displaying earlier and more intense peak serum concentrations. For dose comparison investigations, all SCFAs peaked within 60 min and returned to baseline concentrations by 120 min post-supplementation. A graded dose relationship was present for propionate and butyrate when considering the total circulating exposure across a 240 min monitoring period. In addition, a one-week, twice-daily, repeated supplementation protocol resulted in no changes in basal serum SCFA concentrations. Overall, these data indicate that acetate, propionate, and butyrate display relatively similar circulating concentration profiles following oral co-ingestion, adding knowledge to help inform supplementation strategies for future outcomes where acute elevation of circulating SCFAs is desired.

Intercropping Okra and Castor Bean Reduces Recruitment of Oriental Fruit Moth, Grapholita molesta (Lepidoptera: Tortricidae) in a Pear Orchard.

Intercrops can lower pest densities by increasing plant diversity, altering chemical communication in the arthropod community, and integrating well with other IPM tactics. We used two years of field observations and Y-tube olfactometer assays to explore the effects of intercropping a pear orchard with okra and castor bean on the cosmopolitan fruit-boring pest Grapholita molesta (Lepidoptera: Tortricidae). Intercropping okra reduced G. molesta trap catches in the pear orchard in both years, and intercropping with castor bean reduced them in the second year. Hydrocarbons, phenols, and ketones predominated in the GC-MS assay of okra volatiles, whereas castor bean volatiles were rich in aldehydes, ketones, and esters. Five of the commercially available volatiles released by these plants exhibited repellency to G. molesta in olfactometer trials, especially cinnamaldehyde, dibutyl phthalate, and thymol; the former compound also exhibited attraction to the egg parasitoid Trichogamma dendrolimi (Hymenoptera: Trichogrammatidae). In addition to their repellent properties, okra and castor bean may enhance integrated control of G. molesta in orchards by hosting prey that support populations of generalist predators that either provide biological pest control services within the orchard ecosystem or generate non-consumptive effects that contribute to pest deterence. Among the plant volatiles evaluated, cinnamaldehyde has the best potential for deployment in orchards to repel G. molesta without disrupting augmentative releases of T. dendrolimi.

Synergistic benefits of Funneliformis mosseae and Bacillus paramycoides: Enhancing soil health and soybean tolerance to root rot disease

To explore the response of soil metabolite composition to soybean disease, the effect of the combined inoculation of arbuscular mycorrhizal fungi (AMF) and plant growth-promoting bacteria on soybean root rot caused by Fusarium oxysporum was studied. A factorial completely randomized design with three factors (AMF, Bacillus. paramycoides, and rot disease stress) was conducted, and eight treatments, including normal groups and stress groups, were performed using pot experiments. GC‒MS and enzymatic assays were used to evaluate the soil factors and soybean growth indicators. The results showed that there were significant differences in the composition of metabolites among the different treatment groups, and 23 metabolites were significantly related to soybean biomass. The combined inoculation of Funneliformis mosseae and Bacillus paramycoides resulted in a significant reduction in harmful soil metabolites associated with root rot disease, such as ethylbenzene and styrene. This reduction in metabolites contributed to improving soil health, as evidenced by enhanced soybean defence enzyme activities and microbial activity, and β-1,3-glucanase, chitinase and phenylalanine ammonia-lyase activities were improved to alleviate plant rhizosphere stress. Furthermore, soybean plants inoculated with the synergistic treatments exhibited reduced root rot disease severity and improved growth indicators compared to control plants. Plant height, root dry weight (RDW), and shoot and root fresh weight (SRFW) were improved by 4.18–53.79%, and the AM fungal colonization rate was also improved under stress. The synergistic application of Funneliformis mosseae and Bacillus paramycoides can effectively enhance soil health by inhibiting the production of harmful soil metabolites and improving soybean tolerance to root rot disease. This approach holds promise for the sustainable management of soil-borne diseases in soybean cultivation.

Sitosterol-rich Digera muricata against 7-ketocholesterol and lipopolysaccharide-mediated atherogenic responses by modulating NF-ΚB/iNOS signalling pathway in macrophages.

Digera muricata L., commonly known as Tartara, is an edible herb used as traditional medicine in many countries of Africa and Asia. This study aimed to elucidate the effect of a phytosterol-rich extract of D. muricata on 7-ketocholesterol-mediated atherosclerosis in macrophages. The extract was examined by phytochemical analyses, GC-MS, TLC, DPPH scavenging and hRBC membrane stabilization assays. Macrophage polarization was studied with experimental groups framed based on alamar blue cell viability and griess assays. Regulations of arginase enzyme activity, ROS generation, mitochondrial membrane potential, cell membrane integrity, pinocytosis, lipid uptake and peroxidation, as well as, intracellular calcium deposition were determined. In addition, expressions of atherogenic mediators were analysed using PCR, ELISA and immunocytochemistry techniques. Diverse phytochemicals with higher free radical scavenging activity and anti-inflammatory potential have been detected in the D. muricata. Co-treatment with D. muricata markedly reduced the atherogenic responses induced by 7KCh in the presence of LPS such as ROS, especially, NO and O2- along with lipid peroxidation. Furthermore, D. muricata significantly normalized mitochondrial membrane potential, cell membrane integrity, pinocytic activity, intracellular lipid accumulation and calcium deposition. These results provided us with the potentiality of D. muricata in ameliorating atherogenesis. Additionally, it decreased the expression of pro-atherogenic mediators (iNOS, COX-2, MMP9, IL-6, IL-1β, CD36, CD163 and TGFβ1) and increased anti-atherogenic mediators (MRC1 and PPARγ) with high cellular expressions of NF-κB and iNOS. Results showed the potential of sitosterol-rich D. muricata as a versatile biomedical therapeutic agent against abnormal macrophage polarization and its associated pathologies.

Feasibility study on the homogeneity and stability of megastigmatrienone isomers for the development of certified reference material

Megastigmatrienone is a natural flavoring food additive with a spicy and tobacco-like aroma. A feasibility study on the homogeneity and stability of megastigmatrienone preceded for the development of a certified reference material (CRM). Megastigmatrienone, a mixture of four diastereomers, was prepared using gram-scale synthesis. The purity values of candidate megastigmatrienone reference material (MRM) determined using two independent methods showed comparable results by GC–MS assay (90.89%) and mass balance method (92.42%). Candidate MRM aliquoted into vials was confirmed to be homogenous between the bottles. In the stability test, various packaging methods and environmental conditions capable of stabilizing megastigmatrienone were tested. Candidate MRM aliquoted into vials was unstable from –20 °C to 40 °C for 12 months. However, either antioxidant additive or argon-substituted heat-sealing package significantly improved its stability. These findings suggest that atmospheric reactive radicals may be critical factors resulting in the decomposition of megastigmatrienone. Argon-substituted packaging and storage temperatures below −20 °C are the practical alternatives to sustain its stability. This study systematically demonstrates strategies for stabilizing materials with poor stability. Our findings can be useful for developing megastigmatrienone CRM to guarantee the reliable quality of food additives or foods containing megastigmatrienone.

The Metabolic Profile of Young, Watered Chickpea Plants Can Be Used as a Biomarker to Predict Seed Number under Terminal Drought.

Chickpea is the second-most-cultivated legume globally, with India and Australia being the two largest producers. In both of these locations, the crop is sown on residual summer soil moisture and left to grow on progressively depleting water content, finally maturing under terminal drought conditions. The metabolic profile of plants is commonly, correlatively associated with performance or stress responses, e.g., the accumulation of osmoprotective metabolites during cold stress. In animals and humans, metabolites are also prognostically used to predict the likelihood of an event (usually a disease) before it occurs, e.g., blood cholesterol and heart disease. We sought to discover metabolic biomarkers in chickpea that could be used to predict grain yield traits under terminal drought, from the leaf tissue of young, watered, healthy plants. The metabolic profile (GC-MS and enzyme assays) of field-grown chickpea leaves was analysed over two growing seasons, and then predictive modelling was applied to associate the most strongly correlated metabolites with the final seed number plant-1. Pinitol (negatively), sucrose (negatively) and GABA (positively) were significantly correlated with seed number in both years of study. The feature selection algorithm of the model selected a larger range of metabolites including carbohydrates, sugar alcohols and GABA. The correlation between the predicted seed number and actual seed number was R2 adj = 0.62, demonstrating that the metabolic profile could be used to predict a complex trait with a high degree of accuracy. A previously unknown association between D-pinitol and hundred-kernel weight was also discovered and may provide a single metabolic marker with which to predict large seeded chickpea varieties from new crosses. The use of metabolic biomarkers could be used by breeders to identify superior-performing genotypes before maturity is reached.

Moschus exerted protective activity against H2O2-induced cell injury in PC12 cells through regulating Nrf-2/ARE signaling pathways

The pivotal characteristics of Alzheimer's disease (AD) are irreversible memory loss and progressive cognitive decline, eventually causing death from brain failure. In the various proposed hypotheses of AD, oxidative stress is also regarded as a symbolic pathophysiologic cascade contributing to brain diseases. Using Chinese herbal medicine may be beneficial for treating and preventing AD. As a rare and valuable animal medicine, Moschus possesses antioxidant and antiapoptotic efficacy and is extensively applied for treating unconsciousness, stroke, coma, and cerebrovascular diseases. We aim to evaluate whether Moschus protects PC12 cells from hydrogen peroxide (H2O2)-induced cellular injury. The chemical constituents of Moschus are analyzed by GC-MS assay. The cell viability, reactive oxygen species (ROS) levels, mitochondrial membrane potential (MMP) levels, oxidative stress-related indicators, and apoptotic proteins are determined. Through GC-MS analysis, nineteen active contents were identified. The cell viability loss, lactate dehydrogenase releases, MMP levels, ROS productions, and Malondialdehyde (MDA) activities decreased, and BAX, Caspase-3, and Kelch-like ECH-associated protein 1 expression also significantly down-regulated and heme oxygenase 1, nuclear factor erythroid-2-related factor 2 (Nrf-2), and quinine oxidoreductase 1 expression upregulated after pretreatment of Moschus. The result indicated Moschus has neuroprotective activity in relieving H2O2-induced cellular damage, and the potential mechanism might be associated with regulating the Nrf-2/ARE signaling pathway. A more in-depth and comprehensive understanding of Moschus in the pathogenesis of AD will provide a fundamental basis for in vivo AD animal model research, which may be able to provide further insights and new targets for AD therapy.

Cytotoxic effects of Trachyspermum ammi and Ferula assafoetida on MCF-7 and MDA-MB-468 breast cancer cell lines

Abstract Background The induction of oxidative stress is one of the most important cancer etiologies. Plant essential oils contain many effective antioxidant compounds in improving oxidative stress. In the present study, the pharmacological potential of Trachyspermum ammi essential oil (TAEO) and Ferula assafoetida essential oil (FAEO) was compared in oxidative stress improvement and cytotoxic effect. TAEO and FAEO were prepared by Clevenger apparatus, and the medicinal compounds in the essential oils were evaluated by GC–MS assay. The TAEO and FAEO were also evaluated as to their phenolic and flavonoid content, monovalent reducing power, and total radical scavenging activity, respectively, by Folin–Ciocalteu, aluminum chloride, FRAP, and DPPH methods. The cytotoxic effect of the TAEO and FAEO was evaluated by MTT assay on MCF-7 (ER+) and MDA-MB-468 (ER−) breast cancer cell lines. Results The GC–MS analysis indicated that thymol and (E)-Sec-Butyl propenyl disulfide, respectively, were the highest components of TAEO and FAEO. The phenolic content (P < 0.0001), flavonoid content (P < 0.0001), reducing power (P < 0.0001), radical scavenging activity (P < 0.0001), and cytotoxic effect (P < 0.05) of TAEO were significantly higher than FAEO. The IC50 value of the cytotoxic effect of TAEO on MCF-7 and MDA-MB-468 after 72 h of incubation was, respectively, 192.5 ± 42.57 and 331.4 ± 7.24 ppm. Conclusion The cytotoxic effect of TAEO was more potent on the MCF-7 cell line, probably in an estrogen-dependent manner of cellular growth inhibition. It appears that TAEO has a high capacity for improving oxidative stress and inhibiting cell proliferation in breast cancer.

Analysis of the A549 cell line affected by anticancer bioactive compounds of Actinomycetes isolated from saline soils.

Actinomycetes are filamentous bacteria and the residents of the soil, prone to produce bioactive metabolites. This research aimed to isolate, classify, and investigate the anticancer properties of Actinomycetes secondary metabolites from various saline soils of Qom province. Actinomycetes isolates were molecularly recognized by 16SrRNA gene sequencing after the PCR procedure. The A549 cell line was then exposed to bacterial metabolites to find their cytotoxicity by MTT assay and their capacity to cause apoptosis by Flow cytometry. The expression levels of the bax and bcl-2 genes were determined using Real-time PCR. Bacterial metabolites were distinct by HPLC and GC-MS assays. Sequencing identified three novel Actinomycetes strains, Streptomyces griseoflavus, Streptomyces calvus, and Kitasatospora phosalacineus. The IC50 doses of bacterial metabolites were discovered equal to 1337, 2619, and 4874µg/ml, respectively. Flow cytometric assay revealed that their secondary metabolites were capable of inducing apoptosis in A549 cells by 25%, 14.5%, and 7.58%, respectively. Real-time PCR findings displayed that the bax gene expression in A549 cells treated with S. griseoflavus and S. calvus, comparatively increased (P < 0.0008, P < 0.00056). The expression of the bcl-2 gene was significantly reduced in cells treated with S. griseoflavus and K. phosalacineus (P < 0.0006, P < 0.0004). The findings of this analysis showed the presence of new isolates in a soil sample from Qom province which can produce new anticancer agents and can be considered appropriate candidates for further research to employ as anticancer drugs.

Discovery and therapeutic implications of bioactive dihydroxylated phenolic acids in patients with severe heart disease and conditions associated with inflammation and hypoxia

Our initial studies detected elevated levels of 3,4-dihydroxyphenyllactic acid (DHPLA) in urine samples of patients with severe heart disease when compared with healthy subjects. Given the reported anti-inflammatory properties of DHPLA and related dihydroxylated phenolic acids (DPAs), we embarked on an exploratory multi-centre investigation in patients with no urinary tract infections to establish the possible pathophysiological significance and therapeutic implications of these findings. Chinese and Caucasian patients being treated for severe heart disease or those conditions associated with inflammation (WBC ≥ 10 ×109/L or hsCRP ≥ 3.0 mg/L) and/or hypoxia (PaO2 ≤ 75 mmHg) were enrolled; their urine samples were analyzed by HPLC, HPLC-MS, GC-MS and biotransformation assays. DHPLA was detected in urine samples of patients, but undetectable in healthy volunteers. Dynamic monitoring of inpatients undergoing treatment showed their DHPLA levels declined in proportion to their clinical improvement. In DHPLA-positive patients’ fecal samples, Proteus vulgaris and P. mirabilis were more abundant than healthy volunteers. In culture, these gut bacteria were capable of reversible interconversion between DOPA and DHPLA. Furthermore, porcine and rodent organs were able to metabolize DOPA to DHPLA and related phenolic acids. The elevated levels of DHPLA in these patients suggest bioactive DPAs are generated de novo as part of a human’s defense mechanism against disease. Because DHPLA isolated from Radix Salvia miltiorrhizae has a multitude of pharmacological activities, these data underpin the scientific basis of this medicinal plant’s ethnopharmacological applications as well as highlighting the therapeutic potential of endogenous, natural or synthetic DPAs and their derivatives in humans.

Leaf Extracts of Cistus ladanifer Exhibit Potent Antioxidant and Antiproliferative Activities against Liver, Prostate and Breast Cancer Cells

Chemical composition, antioxidant, and antiproliferative properties of C. ladanifer crude extracts, including hexane (Hex), dichloromethane (DCM), ethyl acetate (E.A) and ethanol (EtOH) were investigated. The chemical composition of C. ladanifer crude extracts was determined by use of GC-MS, whereas DPPH and FRAP assays were employed to determine its antioxidant capacity. The obtained results showed that the ethanolic extract exhibited a significant antioxidant effect recording an IC50 value of 266.6 ± 0.828 μg/mL with DPPH assay, and a higher reducing power 0.494 ± 0.035 using the FRAP test. The extracts exhibited significant antiproliferative activity against three cancer cell lines. The DCM extract exhibited the highest total polyphenol content (76.066 ± 9.978 μg AGE/mg) and was revealed to be more effective against HepG2 (31.54 ± 0.242 μg/mL). The Hex extract that presented the highest flavonoid content (50.209 ± 3.805 μg CE/mg) exhibited the highest antiproliferative activity against 22Rv1 and MDA-MB-231 recording IC50 values 11.32 ± 2.126 μg/mL and 82.4 ± 1.124 μg/mL, respectively. All four extracts exhibited minimal toxicity against human skin-derived fibroblast cells indicating the specificity of their observed anticancer activity. GC-MS analysis identified interesting phytochemicals underlying the obtained antioxidant and cytotoxic activities. Taken together, results of the current study highlight the significance of C. ladanifer as a valuable source of antioxidant and anticancer bioactive compounds, thereby warranting further detailed investigation.

Simple, high-throughput measurement of gut-derived short-chain fatty acids in clinically relevant biofluids using gas chromatography-mass spectrometry

IntroductionThe quantitative measurement of circulating gut bacteria-derived metabolites has increased in recent years due to their associations with health and disease. While much of the previous attention has been placed on metabolites considered as deleterious to health, a shift to the investigation of short-chain fatty acids (SCFAs) as potential health promotors has been observed. ObjectivesTo develop a simple, high-throughput and quantitative assay to measure gut-derived SCFAs in clinically relevant biofluids using gas chromatography-mass spectrometry (GC–MS). MethodsA short (7.5 min) GC–MS assay was optimized for measurement of seven straight- and branched-chain SCFAs and their deuterated isotopes using a wax-based column for analysis without prior derivatization. The assay was validated using routine criteria to assess precision, accuracy, matrix effects, recovery, and extraction reproducibility. Assay applicability was tested in cohorts of healthy individuals and kidney disease patients. ResultsThe assay was demonstrated to be precise, accurate and reproducible with acceptable levels of matrix effect and analyte recovery. Lower limits of detection and quantitation were in the low ng/mL range. An investigation into different blood collection tube chemistries demonstrated that lithium heparin plasma and serum clotting activator tubes are recommended for use in future cross-study comparisons. Kidney disease patient analyses demonstrated variable differences across SCFAs when comparing hemodialysis to earlier stages of chronic kidney disease, demonstrating the suitability of the assay for translation to clinical analyses. ConclusionThe assay has been validated and identified as reliable for use in larger-scale studies for the analysis of SCFAs in human plasma and serum.

Quality-by-design approach for development of aqueous headspace microextraction GC-MS method for targeted metabolomics of small aldehydes in plasma of cardiovascular patients

Lipid peroxidation products, such as short chain aldehydes, are powerful biomarkers of oxidative stress, due to the advantage of long lifetime compared to other metabolites of the lipidome. This work proposes an advanced combined derivatization/solvent-less extraction procedure from plasma followed by rapid Gas Chromatography with Mass Spectrometric detection (GC-MS). A new sample pretreatment protocol is presented which is based on a combination of aldehyde derivatization with methoxyamine under fully aqueous-based conditions of diluted plasma samples followed by headspace solid-phase microextraction (HS-SPME) which is faster compared to methods in the literature serving the same purpose. Being the smallest oximation reagent, methoxyamine derivatization does not require a silylation step of hydroxyl groups as customary and made it possible to have the shortest run times for this series of aldehydes by GC-MS. A Response Surface Methodology (RSM) is employed to optimize the HS-SPME of the aldehyde methoximes to provide insights into the Design Space (DS) of HS-SPME of aldehydes of variable chain lengths and unsaturation. The workflow includes a Quality by Design (QbD) approach for optimization of sample microextraction and derivatization methodology under fully aqueous conditions, in contrast to all reported non-aqueous tedious and long extraction methods in the literature followed by development of a rapid GC-MS assay. The optimal sample preparation obtained from the RSM, and multiple linear regression procedure involved addition of 15 mg methoxyamine (CH3ONH2) and 160 mg Na2SO4 to 0.5 mL plasma diluted to 1 mL with water in an extraction vial followed by HS-SPME using Polydimethylsiloxane/Divinylbenzene fiber at 750 rpm and 77 °C for 15 min. The developed HS-SPME-GC-MS method was validated according to FDA guidelines in SIM mode and applied for targeted determination of lipid peroxidation aldehyde metabolites in plasma of 24 cardiovascular patients vs 20 healthy controls. The run time of the GCMS method was less than 15 min and the LOQ of the 10 targeted aldehydes were 0.5 nM for decanal, 5 nM for hexanal, heptanal, octanal, citronellal and citral, 7 nM for malondialdehyde, 35 nM for 4- hydroxynonenal, 105 nM for 4- hydroxyhexenal and 500 nM for glyoxal. Hexanal, Malondialdehyde and Hydroxynonenal concentrations were significantly higher in patients (p-value<0.05) in the targeted study, while citral was significantly lower as obtained from the untargeted study. Reporting an aldehydic profile signature -whether predictive or diagnostic-for cardiovascular patients would support proper medical intervention at the initiation or progression phases of the disease when expanded on larger number of subjects.

Study on biochemical analysis, antioxidant assay and GCMS analysis of Saccharina japonica

Marine algae are one of the largest sources for biogenic compounds. They produce a wide variety of chemically active metabolites. They are found to possess significant antioxidant, and phytochemical activities. Macroalgae provide a great variety of metabolites and natural bioactive compounds with antimicrobial activity, such as polysaccharides, polyunsaturated fatty acids, phlorotannins other phenolic compounds and carotenoids. In the present study the phytochemical, amylase activity, GCMS and Antioxidant assays such as ABTS assay and DPPH assays were carried out for Saccharina japonica using two different solvents, petroleum ether and ethyl acetate. For antioxidant activity, DPPH radical assay and ABTS radical cation scavenging assay were carried out.

Phytochemical profiles, antioxidant and antimicrobial activity of Actinidia polygama and A. arguta fruits and leaves

Plants of two species of Actinidia genus grown in an adverse steppe climate were examined in terms of secondary metabolites’ accumulation, antioxidant potential, and antimicrobial ability. The aim of the work was to reveal whether the introduced plants A. arguta and A. polygama retain their well-known health benefits. Total content of polyphenols (549.2 and 428.1 mg GAE/100 g FW, respectively), flavonoids, and phenolic acids as well as total antioxidant activity and reducing power of the fruit isopropanol extracts were found to be equal or even higher than the reported data on kiwifruit varieties cultivated in China and other regions. Antioxidant potential and phenolic compounds’ content in the fruit peel of both species were higher when compared to pulp, while corresponding indices of leaves exceeded those of the fruit. Disc-diffusion assays showed low to moderate antibacterial activity of A. arguta and A. polygama fruit and leaf extracts against collection Gram-negative and Gram-positive strains. Clinical strains of P. aeruginosa and E. coli resistant to the action of ofloxacin were notably inhibited by A. arguta and A. polygama fruit and leaf crude extracts. Inhibiting effects of plant extracts on clinical strains of K. pneumoniae and A. baumannii were comparable with the effect of ofloxacin. GC-MS assays identified 23 and 36 chemical constituents, respectively in A. arguta and A. polygama fruit isopropanol extracts. The main compounds in both extracts were 2-propenoic acid, pentadecyl ester followed by squalene, 7,9-di-tert-butyl-1-oxaspiro(4,5)deca-6,9-dien-2,8-dione, octadecanoic acid, 2-oxo-methyl ester, ethyl-isoallocholate, and phytol having known bioactivities. Our findings confirmed the preservation of useful properties by the introduced plants and also indicated the rich health-promoting abilities and expedience of cultivating A. arguta and A. polygama in a steppe climate.

Identification and Determination of 1,3-Thiazinane-4-carboxylic Acid in Human Urine-Chromatographic Studies.

It is well established that homocysteine (Hcy) and its thiolactone (HTL) are reactive towards aldehydes in an aqueous environment, forming substituted thiazinane carboxylic acids. This report provides evidence that Hcy/HTL and formaldehyde (FA) adduct, namely 1,3-thiazinane-4-carboxylic acid (TCA) is formed in vivo in humans. In order to provide definitive proof, a gas chromatography–mass spectrometry (GC–MS) based method was elaborated to identify and quantify TCA in human urine. The GC–MS assay involves chemical derivatization with isobutyl chloroformate (IBCF) in the presence of pyridine as a catalyst, followed by an ethyl acetate extraction of the obtained isobutyl derivative of TCA (TCA-IBCF). The validity of the method has been demonstrated based upon United States Food and Drug Administration recommendations. The assay linearity was observed within a 1–50 µmol L−1 range for TCA in urine, while the lowest concentration on the calibration curve was recognized as the limit of quantification (LOQ). Importantly, the method was successfully applied to urine samples delivered by apparently healthy volunteers (n = 15). The GC–MS assay may provide a new analytical tool for routine clinical analysis of the role of TCA in living systems in the near future.