Mass Spectrometry-based Metabolomics Approach Research Articles

A Comprehensive Multi-Omics Study of Serum Alterations in Red Deer Infected by the Liver Fluke Fascioloides magna

Liver fluke infections are acknowledged as diseases with global prevalence and significant implications for both veterinary and public health. The large American liver fluke, Fascioloides magna, is a significant non-native parasite introduced to Europe, threatening the survival of local wildlife populations. The aim of this study was to analyze differences in the serum proteome and metabolome between F. magna-infected and control red deer. Serum samples from red deer were collected immediately following regular hunting operations, including 10 samples with confirmed F. magna infection and 10 samples from healthy red deer. A proteomics analysis of the serum samples was performed using a tandem mass tag (TMT)-based quantitative approach, and a metabolomics analysis of the serum was performed using an untargeted mass spectrometry-based metabolomics approach. A knowledge-driven approach was applied to integrate omics data. Our findings demonstrated that infection with liver fluke was associated with changes in amino acid metabolism, energy metabolism, lipid metabolism, inflammatory host response, and related biochemical pathways. This study offers a comprehensive overview of the serum proteome and metabolome in response to F. magna infection in red deer, unveiling new potential targets for future research. The identification of proteins, metabolites, and related biological pathways enhances our understanding of host–parasite interactions and may improve current tools for more effective liver fluke control.

Integrated analysis of metabolome and microbiome in a rat model of perimenopausal syndrome.

In this work, we employed 16S ribosomal RNA gene sequencing to analyze the gut microbes in stool samples. In addition, we conducted an ultra-high-performance liquid chromatography-tandem mass spectrometry-based metabolomics approach on gut tissue and serum obtained from rats with perimenopausal syndrome (PMS) and healthy controls. By characterizing the composition and metabolomic properties of gut microbes in PMS rats, we aim to enhance our understanding of their role in women's health, emphasizing the significance of regulating gut microbes in the context of menopausal women's well-being. We aim to provide a theoretical basis for the prevention and treatment of PMS in terms of gut microflora as well as metabolism.

Comparative analysis and evaluation of wild and cultivated Radix Fici Simplicissimae using an UHPLC-Q-Orbitrap mass spectrometry-based metabolomics approach

Radix Fici Simplicissimae (RFS) is widely studied, and is in demand for its value in medicines and food products, with increased scientific focus on its cultivation and breeding. We used ultra-high-performance liquid chromatography quadrupole-orbitrap mass spectrometry-based metabolomics to elucidate the similarities and differences in phytochemical compositions of wild Radix Fici Simplicissimae (WRFS) and cultivated Radix Fici Simplicissimae (CRFS). Untargeted metabolomic analysis was performed with multivariate statistical analysis and heat maps to identify the differences. Eighty one compounds were identified from WRFS and CRFS samples. Principal component analysis and orthogonal partial least squares discrimination analysis indicated that mass spectrometry could effectively distinguish WRFS from CRFS. Among these, 17 potential biomarkers with high metabolic contents could distinguish between the two varieties, including seven phenylpropanoids, three flavonoids, one flavonol, one alkaloid, one glycoside, and four organic acids. Notably, psoralen, apigenin, and bergapten, essential metabolites that play a substantial pharmacological role in RFS, are upregulated in WRFS. WRFS and CRFS are rich in phytochemicals and are similar in terms of the compounds they contain. These findings highlight the effects of different growth environments and drug varieties on secondary metabolite compositions and provide support for targeted breeding for improved CRFS varieties.

Mass spectrometry-based metabolomics for the elucidation of alkaloid biosynthesis and function in invasive Vincetoxicum rossicum populations

The genus Vincetoxicum includes a couple of highly invasive vines in North America that threaten biodiversity and challenge land management strategies. Vincetoxicum species are known to produce bioactive phenanthroindolizidine alkaloids that might play a role in the invasiveness of these plants via chemical interactions with other organisms. Untargeted, high-resolution mass spectrometry-based metabolomics approaches were used to explore specialized metabolism in Vincetoxicum plants collected from invaded sites in Ontario, Canada. All metabolites corresponding to alkaloids in lab and field samples of V. rossicum and V. nigrum were identified, which collectively contained 25 different alkaloidal features. The biosynthesis of these alkaloids was investigated by the incorporation of the stable isotope-labelled phenylalanine precursor providing a basis for an updated biosynthetic pathway accounting for the rapid generation of chemical diversity in invasive Vincetoxicum. Aqueous extracts of aerial Vincetoxicum rossicum foliage had phytotoxic activity against seedlings of several species, resulting in identification of tylophorine as a phytotoxin; tylophorine and 14 other alkaloids from Vincetoxicum accumulated in soils associated with full-sun and a high-density of V. rossicum. Using desorption-electrospray ionization mass spectrometry, 15 alkaloids were found to accumulate at wounded sites of V. rossicum leaves, a chemical cocktail that would be encountered by feeding herbivores. Understanding the specialized metabolism of V. rossicum provides insight into the roles and influences of phenanthroindolizidine alkaloids in ecological systems and enables potential, natural product-based approaches for the control of invasive Vincetoxicum and other weedy species.

Dulaglutide treatment reverses depression-like behavior and hippocampal metabolomic homeostasis in mice exposed to chronic mild stress.

Treatment strategies for depression based on interventions for glucose and lipid metabolism disorders are receiving increasing attention. Investigating the mechanism of their antidepressant effect and exploring new diagnostic and therapeutic biomarkers have attracted increasing attention. Dulaglutide, a long-acting GLP-1 receptor agonist, has been reported to alleviate cognitive deficits and neuronal damage. However, the antidepressant effect of dulaglutide and, especially, the underlying mechanism are still poorly understood. In this study, we aimed to explore the underlying biomarkers of depression and potential modulatory targets of dulaglutide in chronic mild stress (CMS) mice. Sixty mice were randomly divided into a control group (CON group), a CMS+Vehicle group (CMS+Veh group), a CMS+0.3mg/kg dulaglutide group (Low Dula group), and a CMS+0.6mg/kg dulaglutide group (High Dula group). Numerous behavioral tests, mainly the open field test, forced swimming test, and tail suspension test, were applied to evaluate the potential effect of dulaglutide treatment on anxiety- and depression-like behaviors in mice exposed to chronic stress. Furthermore, a liquid chromatography-tandem mass spectrometry-based metabolomics approach was utilized to investigate the associated mechanisms of dulaglutide treatment. Three weeks of dulaglutide treatment significantly reversed depressive-like but not anxiety-like behaviors in mice exposed to chronic stress for 4 weeks. The results from the metabolomics analysis showed that a total of 20 differentially expressed metabolites were identified between the CON and CMS+Veh groups, and 46 metabolites were selected between the CMS+Veh and High Dula groups in the hippocampus of the mice. Comprehensive analysis indicated that lipid metabolism, amino acid metabolism, energy metabolism, and tryptophan metabolism were disrupted in model mice that experienced depression and underwent dulaglutide therapy. The antidepressant effects of dulaglutide in a CMS depression model were confirmed. We identified 64 different metabolites and four major pathways associated with metabolic pathophysiological processes. These primary data provide a new perspective for understanding the antidepressant-like effects of dulaglutide and may facilitate the use of dulaglutide as a potential therapeutic strategy for depression.

Altered Gut Microbiota and Short-chain Fatty Acids in Chinese Children with Constipated Autism Spectrum Disorder

Gastrointestinal symptoms are more prevalent in children with autism spectrum disorder (ASD) than in typically developing (TD) children. Constipation is a significant gastrointestinal comorbidity of ASD, but the associations among constipated autism spectrum disorder (C-ASD), microbiota and short-chain fatty acids (SCFAs) are still debated. We enrolled 80 children, divided into the C-ASD group (n = 40) and the TD group (n = 40). In this study, an integrated 16S rRNA gene sequencing and gas chromatography–mass spectrometry-based metabolomics approach was applied to explore the association of the gut microbiota and SCFAs in C-ASD children in China. The community diversity estimated by the Observe, Chao1, and ACE indices was significantly lower in the C-ASD group than in the TD group. We observed that Ruminococcaceae_UCG_002, Erysipelotrichaceae_UCG_003, Phascolarctobacterium, Megamonas, Ruminiclostridium_5, Parabacteroides, Prevotella_2, Fusobacterium, and Prevotella_9 were enriched in the C-ASD group, and Anaerostipes, Lactobacillus, Ruminococcus_gnavus_group, Lachnospiraceae_NK4A136_group, Ralstonia, Eubacterium_eligens_group, and Ruminococcus_1 were enriched in the TD group. The propionate levels, which were higher in the C-ASD group, were negatively correlated with the abundance of Lactobacillus taxa, but were positively correlated with the severity of ASD symptoms. The random forest model, based on the 16 representative discriminant genera, achieved a high accuracy (AUC = 0.924). In conclusion, we found that C-ASD is related to altered gut microbiota and SCFAs, especially decreased abundance of Lactobacillus and excessive propionate in faeces, which provide new clues to understand C-ASD and biomarkers for the diagnosis and potential strategies for treatment of the disorder. This study was registered in the Chinese Clinical Trial Registry (www.chictr.org.cn; trial registration number ChiCTR2100052106; date of registration: October 17, 2021).

Mass spectrometry-based untargeted metabolomics approaches for comprehensive structural annotation of bioactive metabolites from bushy cashew (Anacardium humile) fruits

Anacardium humile (bushy cashew) is a native Brazilian plant with substantial pharmacological potential and noteworthy commercial significance in the food industry. This study introduces an untargeted metabolomics approach based on mass spectrometry for the comprehensive structural annotation of bioactive metabolites. The fruits were collected from three distinct sites and subjected to LC-HRMS/MS analysis. A total of eighty-eight compounds were putatively annotated across various metabolite classes. This unveiled a metabolic profile characterized by notable concentrations of polyphenols, including flavonoids, tannins, phenolic acids, and quinones, while aliphatic acids and terpenes were found in limited quantities. Noteworthy, no significant disparities in the metabolic content were observed among the collection sites. The three principal metabolites (peonidin 3-O-glucopyranoside, methylcyanidin and methyldelphinidin) underwent assessment for antioxidant activity via molecular docking analysis, subsequently generating structure-based feature pharmacophores. The process of annotation propagation yielded a comprehensive qualitative appraisal of the A. humile metabolome. The outcomes obtained offer potential candidates for further exploration of their nutraceutical attributes.

Unlocking Potentially Therapeutic Phytochemicals in Capadulla (Doliocarpus dentatus) from Guyana Using Untargeted Mass Spectrometry-Based Metabolomics.

Doliocarpus dentatus is thought to have a wide variety of therapeutic phytochemicals that allegedly improve libido and cure impotence. Although a few biomarkers have been identified with potential antinociceptive and cytotoxic properties, an untargeted mass spectrometry-based metabolomics approach has never been undertaken to identify therapeutic biofingerprints for conditions, such as erectile dysfunction, in men. This study executes a preliminary phytochemical screening of the woody vine of two ecotypes of D. dentatus with renowned differences in therapeutic potential for erectile dysfunction. Liquid chromatography-mass spectrometry-based metabolomics was used to screen for flavonoids, terpenoids, and other chemical classes found to contrast between red and white ecotypes. Among the metabolite chemodiversity found in the ecotype screens, using a combination of GNPS, MS-DIAL, and SIRIUS, approximately 847 compounds were annotated at levels 2 to 4, with the majority of compounds falling under lipid and lipid-like molecules, benzenoids and phenylpropanoids, and polyketides, indicative of the contributions of the flavonoid, shikimic acid, and terpenoid biosynthesis pathways. Despite the extensive annotation, we report on 138 tentative compound identifications of potentially therapeutic compounds, with 55 selected compounds at a level-2 annotation, and 22 statistically significant therapeutic biomarkers, the majority of which were polyphenols. Epicatechin methyl gallate, catechin gallate, and proanthocyanidin A2 had the greatest significant differences and were also relatively abundant among the red and white ecotypes. These putatively identified compounds reportedly act as antioxidants, neutralizing damaging free radicals, and lowering cell oxidative stress, thus aiding in potentially preventing cellular damage and promoting overall well-being, especially for treating erectile dysfunction (ED).

Evolutionary metabolomics of specialized metabolism diversification in the genus Nicotiana highlights N-acylnornicotine innovations.

Specialized metabolite (SM) diversification is a core process to plants' adaptation to diverse ecological niches. Here, we implemented a computational mass spectrometry-based metabolomics approach to exploring SM diversification in tissues of 20 species covering Nicotiana phylogenetics sections. To markedly increase metabolite annotation, we created a large in silico fragmentation database, comprising >1 million structures, and scripts for connecting class prediction to consensus substructures. Together, the approach provides an unprecedented cartography of SM diversity and section-specific innovations in this genus. As a case study and in combination with nuclear magnetic resonance and mass spectrometry imaging, we explored the distribution of N-acylnornicotines, alkaloids predicted to be specific to Repandae allopolyploids, and revealed their prevalence in the genus, albeit at much lower magnitude, as well as a greater structural diversity than previously thought. Together, the data integration approaches provided here should act as a resource for future research in plant SM evolution.

Nutritional and Metabolomic Changes of Juvenile Farmed Abalone (Haliotis iris) in New Zealand

Seasonal variations play a crucial role in the physiology, immune responses, and nutritional profile of aquatic animals. Unpredictable water temperature fluctuations, especially those caused by climate change, may negatively affect feed consumption and growth of cultured organisms, such as abalone. In addition, metabolic and nutritional changes across different seasons may have significant effects on aquaculture production. This study aimed to investigate biochemical and metabolic alterations in healthy abalone (Haliotis iris) during 1 year of grow out in a land-based farm in New Zealand. Proximate analyses were used to identify nutritional variations in whole animal tissues, and a gas chromatography–mass spectrometry-based metabolomics approach was used to identify metabolic changes in adductor muscle of abalone during different seasons in the 1-year sampling period. Results showed that protein content was higher in warmer months compared with colder months, whereas lipid, ash, and carbohydrate contents remained generally constant throughout the year. Metabolic profile fluctuations indicated higher amounts of glutamic acid, glutathione, methionine, lysine, serine, tyrosine, and glycine in January and March compared with October and July, indicating possible amino acid breakdown and collagen degradation due to warmer temperatures. Although the proximate analyses findings revealed no signs of nutritional deficiencies in abalone among seasons, the metabolic profiles suggested possible thermal stress during summer months. This study provides a foundation for further nutritional studies to optimise seasonal diets for farmed Haliotis iris and highlights the need to monitor thermal stress effects, especially during summer and/or heatwave events.

Light-Induced Changes in Secondary Metabolite Production of Trichoderma atroviride.

Many studies aim at maximizing fungal secondary metabolite production but the influence of light during cultivation has often been neglected. Here, we combined an untargeted isotope-assisted liquid chromatography-high-resolution mass spectrometry-based metabolomics approach with standardized cultivation of Trichoderma atroviride under three defined light regimes (darkness (PD), reduced light (RL) exposure, and 12/12 h light/dark cycle (LD)) to systematically determine the effect of light on secondary metabolite production. Comparative analyses revealed a similar metabolite profile upon cultivation in PD and RL, whereas LD treatment had an inhibiting effect on both the number and abundance of metabolites. Additionally, the spatial distribution of the detected metabolites for PD and RL was analyzed. From the more than 500 detected metabolites, only 25 were exclusively produced upon fungal growth in darkness and 85 were significantly more abundant in darkness. The majority were detected under both cultivation conditions and annotation revealed a cluster of substances whose production followed the pattern observed for the well-known T. atroviride metabolite 6-pentyl-alpha-pyrone. We conclude that cultivation of T. atroviride under RL can be used to maximize secondary metabolite production.

Untargeted mass spectrometry-based metabolomics approach unveils biochemical changes in compound probiotic fermented milk during fermentation

Probiotic functional products have drawn wide attention because of their increasing popularity. However, few studies have analyzed probiotic-specific metabolism in the fermentation process. This study applied UPLC-QE-MS-based metabolomics to track changes in the milk metabolomes in the course of fermentation by two probiotic strains, Lacticaseibacillus paracasei PC-01 and Bifidobacterium adolescentis B8589. We observed substantial changes in the probiotic fermented milk metabolome between 0 and 36 h of fermentation, and the differences between the milk metabolomes at the interim period (36 h and 60 h) and the ripening stage (60 h and 72 h) were less obvious. A number of time point-specific differential metabolites were identified, mainly belonging to organic acids, amino acids, and fatty acids. Nine of the identified differential metabolites are linked to the tricarboxylic acid cycle, glutamate metabolism, and fatty acid metabolism. The contents of pyruvic acid, γ-aminobutyric acid, and capric acid increased at the end of fermentation, which can contribute to the nutritional quality and functional properties of the probiotic fermented milk. This time-course metabolomics study analyzed probiotic-specific fermentative changes in milk, providing detailed information of probiotic metabolism in a milk matrix and the potential beneficial mechanism of probiotic fermented milk.

Authentication of Tinospora cordifolia derived herbal supplements using high resolution mass spectrometry-based metabolomics approach – A pilot study

Tinospora cordifolia herbal supplements have recently gained prominence due to their promising immunomodulatory and anti-viral effects against SARS-CoV-2. Mislabelling or diluting Tinospora supplements for profit may harm public health. Thus, validating the label claim of these supplements in markets is critical. This study investigated how high resolution mass spectrometry-based metabolomics and chemometrics can be used to distinguish Tinospora cordifolia from two other closely related species (T. crispa and T. sinensis). The Orthogonal Partial Least Square Discriminant Analysis (OPLS-DA) and PLS-DA based chemometric models predicted the species identity of Tinospora with 94.44% accuracy. These classification models were trained using 54 T. cordifolia, 21 T. crispa, and 21 T. sinensis samples. We identified 7 biomarkers, including corydine, malabarolide, ecdysterone, and reticuline, which discriminated Tinospora cordifolia from the two other species. The label claim of 25 commercial Tinospora samples collected from different parts of India was verified based on the relative abundance of the biomarker compounds, of which 20 were found authentic. The relative abundance of biomarkers significantly varied in the 5 suspicious market samples. This pilot study demonstrates a robust metabolomic approach for authenticating Tinospora species, which can further be used in other herbal matrices for product authentication and securing quality.

Effects of extended transition milk feeding on blood metabolites of female Holstein dairy calves at 3 weeks of age: a liquid chromatography with tandem mass spectrometry-based metabolomics approach

Transition milk (TRM) is a rich source of bioactive components that promotes intestinal development and growth, and reduces the susceptibility to diarrhoea in calves. The objective of this study was to characterise the effects of replacing pasteurised waste milk (none-saleable milk containing antibiotic and/or drug residues) with pasteurised TRM for 3 wk on blood metabolites of dairy calves at 21 d of age. A total of 84 healthy newborn female Holstein calves was blocked by birth order and assigned randomly to four treatment groups with partial replacement of pasteurised waste milk by TRM (second milking after parturition) at 0 (0 L/day TRM + 6 L/day milk), 0.5 (0.5 L/day TRM + 5.5 L/day milk), 1 (1 L/day TRM + 5 L/day milk), or 2 L (2 L/day TRM + 4 L/day milk) for a 21-day period. Serum metabolome was determined by liquid chromatography with tandem mass spectrometry-based metabolomics analysis on a subset of 26 randomly selected individuals from calves fed pasteurised waste milk (CON, 6 L/d milk; n = 13) or TRM (2 L/d TRM + 4 L/d milk; n = 13) at 21 d of age. The identified metabolites (194 out of 265) were categorised according to chemical class and the number of metabolites per class in the serum, amongst which glycerophospholipids 16% (n = 43), fatty acyls 7% (n = 19), organic acids 7% (n = 18), organic heterocyclic compounds 5% (n = 13), benzenoids 5% (n = 12), sphingolipids 5% (n = 12), organic oxygen compounds 4% (n = 11), and nucleic acids 3% (n = 9), were the predominant types. Significant differences in metabolites were determined by the volcano plot. Applying the volcano plot, only two metabolites (ceramide and phosphatidylserine) were significantly different between CON and TRM. Overall, our results suggested that prolonged TRM feeding for 3 wk had little effect on the serum metabolome of the dairy calves. We speculate that the potential effects of feeding TRM for 3 wk compared with waste milk were spatially limited to affect the composition of the local gut microbial community and the growth or function of the intestinal epithelium, not allowing detection of the likely effects in the serum through a metabolomic approach.

Identifying the intervention mechanisms of polydatin in hyperuricemia model rats by using UHPLC-Q-Exactive Orbitrap mass spectroscopy metabonomic approach.

Polydatin is a biologically active compound found in mulberries, grapes, and Polygonum cuspidatum, and it has uric acid-lowering effects. However, its urate-lowering effects and the molecular mechanisms underlying its function require further study. In this study, a hyperuricemic rat model was established to assess the effects of polydatin on uric acid levels. The body weight, serum biochemical indicators, and histopathological parameters of the rats were evaluated. A UHPLC-Q-Exactive Orbitrap mass spectrometry-based metabolomics approach was applied to explore the potential mechanisms of action after polydatin treatment. The results showed a trend of recovery in biochemical indicators after polydatin administration. In addition, polydatin could alleviate damage to the liver and kidneys. Untargeted metabolomics analysis revealed clear differences between hyperuricemic rats and the control group. Fourteen potential biomarkers were identified in the model group using principal component analysis and orthogonal partial least squares discriminant analysis. These differential metabolites are involved in amino acid, lipid, and energy metabolism. Of all the metabolites, the levels of L-phenylalanine, L-leucine, O-butanoylcarnitine, and dihydroxyacetone phosphate decreased, and the levels of L-tyrosine, sphinganine, and phytosphingosine significantly increased in hyperuricemic rats. After the administration of polydatin, the 14 differential metabolites could be inverted to varying degrees by regulating the perturbed metabolic pathway. This study has the potential to enhance our understanding of the mechanisms of hyperuricemia and demonstrate that polydatin is a promising potential adjuvant for lowering uric acid levels and alleviating hyperuricemia-related diseases.

Changes in Physicochemical Properties, Metabolites and Antioxidant Activity of Edible Grass during Spontaneous Fermentation

Fermentation is a crucial technology to improve the nutritional and functional properties of food materials. In this study, edible grass was processed by spontaneous fermentation. Changes in physicochemical properties, metabolites, and antioxidant activity of edible grass were investigated by colorimetric method and chromatography mass spectrometry-based non-targeted metabolomics approach during fermentation. The highest total polyphenol and total flavonoid contents, and free radical scavenging abilities were observed on the 17th day of fermentation. The maximum activity of superoxide dismutase was maintained stable in the fermentation time range of 7–70 days. In total, 16 differential metabolites were identified with fermentation duration up to 124 days. Fermented edible grass exerted protection from H2O2-induced cytotoxicity on HepG2 cells, regulating by the reduction in reactive oxygen species level and the increase in antioxidant enzyme activities. Overall, this study confirms that fermented edible grass obtained by spontaneous fermentation presented favorable nutritional and functional quality, and is expected to be a kind of food with antioxidant function.

Micafungin effect on Pseudomonas aeruginosa metabolome, virulence and biofilm: potential quorum sensing inhibitor

The prevalence of antibiotic resistance in Pseudomonas aeruginosa places a heavy burden on the health care sectors urging the need to find alternative, non-antibiotic strategies. The interference with the P. aeruginosa quorum sensing (QS) system represents a promising alternative strategy to attenuate the bacterial virulency and its ability to form biofilms. Micafungin has been reported to impede the pseudomonal biofilm formation. However, the influences of micafungin on the biochemical composition and metabolites levels of P. aeruginosa have not been explored. In this study, the effect of micafungin (100 µg/mL) on the virulence factors, QS signal molecules and the metabolome of P. aeruginosa was studied using exofactor assay and mass spectrometry-based metabolomics approaches. Furthermore, confocal laser scanning microscopy (CLSM) using the fluorescent dyes ConA-FITC and SYPRO® Ruby was used to visualize micafungin disturbing effects on the pseudomonal glycocalyx and protein biofilm-constituents, respectively. Our findings showed that micafungin significantly decreased the production of various QS-controlled virulence factors (pyocyanin, pyoverdine, pyochelin and rhamnolipid), along with a dysregulation in the level of various metabolites involved in QS system, lysine degradation, tryptophan biosynthesis, TCA cycle, and biotin metabolism. In addition, the CLSM examination showed an altered matrix distribution. The presented findings highlight the promising role of micafungin as a potential quorum sensing inhibitor (QSI) and anti-biofilm agent to attenuate P. aeruginosa pathogenicity. In addition, they point to the promising role of metabolomics study in investigating the altered biochemical pathways in P. aeruginosa.

Mass spectrometry-based metabolomics approach and in vitro assays revealed promising role of 2,3-dihydroquinazolin-4(1H)-one derivatives against colorectal cancer cell lines

Colorectal cancer (CRC) is the most frequent form of gastrointestinal cancer and one of the major causes of human mortality worldwide. Many of the current CRC therapies have limitations due to multidrug resistance and/or severe side effects. Quinazoline derivatives are promising lead compounds with a wide range of pharmacological actions. In this study, the effect of seven synthesized 2,3-dihydroquinazolin-4(1H)-one analogues as potential anticancer agents against two CRC cell lines (HCT116 and SW480) was investigated using cell viability proliferation, migration, adhesion and invasion assays. A liquid chromatography-mass spectrometry (LC-MS/MS) metabolomics approach was used to identify the underlying biochemical pathways disturbed in treated-HCT116 cells. Cell viability proliferation assay revealed that four compounds (C2, C3, C5, and C7) had IC50 < 10 µM with C5 displaying the most potent cytotoxic effect (IC50 1.4 and 0.3 µM against HCT116 and SW480, respectively). Additionally, the compounds showed suppression of wound closure after 72 h, and both C2 and C5 significantly decreased the number of adherent cells and suppressed HCT116 cells invasion. Metabolomics study revealed that C5 induced significant perturbations in the level of several metabolites including spermine, polyamines, glutamine, creatine and carnitine, and altered biochemical processes essential for cell proliferation and progression such as amino acids biosynthesis and metabolism, redox homeostasis, energy related processes (e.g., fatty acid oxidation, second Warburg like effect) and one-carbon metabolism. Our findings indicate that 2,3-dihydroquinazolin-4(1H)-one analogues, particularly C5, have promising anticancer properties, and shed light on the role of metabolomics in identifying new therapeutic targets and providing better understanding of the pathways altered in treated cancer cells.

A liquid chromatography-mass spectrometry-based metabolomics strategy to explore plant metabolic diversity.

Plants are expert chemists producing millions of metabolites, only a fraction of which are known to date. Plant metabolomics explores the rationale for highly diverse metabolites evolved and synthesized by plants. Over two-thirds of modern medicines are somehow inspired and/or derived from plants, making the identification of phytochemicals a means of discovering new medicines to challenge existing and emerging diseases. This chapter introduces our established liquid chromatography-tandem mass spectrometry-based untargeted metabolomics approach centered around discovering specialized metabolites (so-called secondary metabolites) across broad lineages of nonmodel plant species. Detecting hundreds to thousands of metabolite peaks, including assigning chemical identity, makes metabolomics data generation and analysis a very complex process. Various mass spectrometry techniques are currently being developed to approach the comprehensive metabolome. Among them, untargeted metabolomics can provide new biological insights by simultaneously and unbiasedly measuring and analyzing all detected metabolites. We have provided a hands-on modular account for untargeted plant metabolomics, from preparing plant biological samples to data analysis and processing using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. The methods described here offer a foundation and expert opinion on plant metabolome analysis.

Untargeted metabolomics reveals the combination effects and mechanisms of Huangqi-fuzi herb-pair against doxorubicin-induced cardiotoxicity

Ethnopharmacological relevanceQifu decoction (QFD) is a famous traditional Chinese medicine (TCM) composed of Astragali Radix (HuangQi) and Aconiti Lateralis Radix Praeparaia (Fuzi), which can alleviate doxorubicin (DOX)-induced cardiotoxicity (DIC). However, its protective mechanism remains obscured. Aim of the studyThe present study aimed to uncover the cardioprotective mechanism and the synergistic effect of QFD against DIC in mice. Materials and methodsThe cardioprotective activity of QFD against DIC was assessed by electrocardiogram, serum biochemical assays and histopathology. Mass spectrometry-based metabolomic approach was conducted to elucidate the preventive mechanisms of QFD, HuangQi decoction (HQD), and Fuzi decoction (FZD) against DIC. QFD, HQD, FZD-targeted metabolic pathways were identified and compared to investigate the synergistic mechanism of QFD by computational systems analysis. Quantitative real-time PCR (qRT-PCR) was further employed to validate the key metabolic pathways at the level of the gene. ResultsThe electrocardiogram combined with the biochemical analysis and histopathology showed that the protection effects were sorted as QFD > HQD ≈ FZD. A total of 41 metabolites contributing to DIC were identified in the mice serum, among which 32, 12 and 10 metabolites were significantly reverted by QFD, HQD and FZD, respectively. Metabolic pathway analysis revealed that DOX perturbed 12 metabolic pathways, and QFD, HQD, and FZD-treated groups could significantly reverse 12, 7 and 6 metabolic pathways of these 12 metabolic pathways. Metabolic pathway and qRT-PCR revealed that QFD could protect DIC mainly by regulating energy metabolism, amino acids metabolism, arachidonic acid metabolism and glycerophospholipid metabolism, and HQD and FZD mutually reinforced each other. ConclusionThese evidences revealed that QFD was a promising drug candidate for DIC by maintaining metabolic homeostasis. Meanwhile, this work provided a useful approach for evaluating the efficacy and the synergistic effects of TCMs against cardiomyopathy.