Untargeted Metabolite Profiling Research Articles

Untargeted Metabolomics and Gut Microbiota Modulation Study of Fermented Brown Rice for Obesity.

Obesity or excess adipose tissue mass increases the risk of heart disease, hypertension, and diabetes. Obesity might be prevented by consuming plant-based probiotic fermented foods. This study aimed to determine whether adding Pediococcus acidilactici MNL5 to fermented brown rice (FBR) enhances its metabolites, lipase activity, and antioxidant efficiency. UHPLC-Q-TOF-MS/MS analysis revealed significant changes in untargeted metabolite profiles, while, compared with those of raw brown rice (RBR), FBR contained more antioxidant and lipase inhibitors. We evaluated the FBR in HFD (high-fat-diet)-induced obese mice by employing biochemical, histological, gut microbiome, and serum metabolomics approaches. FBR MD (250 mg/kg) decreased body weight (BW) and fat content compared with RBR. With subsequent FBR MD, mice fed a HFD may have reduced serum lipid levels. A HFD with a mid-dose FBR improved the gut microbiota diversity, composition, and structure; reduced the abundance of obesity-related genera such as Helicobacter, Clostridium, and Desulfovibrio; and promoted the abundance of beneficial genera such as Bifidobacterium, Akkermansia, and Lactobacillus, which are inversely correlated with BW, total cholesterol, TG, LDL-C, and HDL-C. In addition, FBR MD has been associated with increased levels of palmitic acid, EPA, oleic acid, α-linolenic acid, indole, dodecanoic acid, and amino acids. FBR, in its entirety, has exhibited promise as a functional material for ameliorating obesity.

Insights into seed coats of nine cultivars of Australian lupin: Unravelling LC-QTOF MS-based biochemical profiles, nutritional, functional, antioxidant, and antidiabetic properties together with rationalizing antidiabetic mechanism by in silico approaches

Lupins, and other legumes, have attained international interest due to their reported remarkable health benefits. Currently, the seed coats are discarded as waste or animal feed. The research presented here summarizes the potential for incorporating the seed coats into ‘whole grain’ foods. We aimed to identify metabolites found in the seed coats of nine commercial Australian cultivars of lupin (Lupinus angustifolius and L. albus species), and to evaluate and compare their functional, nutritional, antioxidant, and antidiabetic properties, along with in silico exploration of mechanisms of action for selected identified secondary metabolites. The seed coats were found to contain 79 to 90% dietary fibers and substantial quantity of essential macrometals. LC-QTOF MS-based, untargeted bioactive metabolite profiling explored a total of 673 chemical entities, and identified 63 bioactive secondary metabolites including: biophenols, unsaturated fatty acids, triterpenoids, alkaloids, and dietary prebiotics (insoluble fibers). The seed coats from these nine cultivars show substantial antioxidant activity. The cultivars of L. angustifolius inhibit α-amylase and α-glucosidase significantly in vitro. Moreover, in silico docking and dynamic simulation along with ADME/T analysis suggest that quercetin 3-methyl ether and 8-C-methylquercetin 3-methyl ether as molecules, novel in lupin seed coats, are responsible for the α-amylase and α-glucosidase inhibition. The findings indicated that lupin seed coats might be beneficial food components, rather than be discarded as ‘mill waste’

Combined transcriptome and metabolome analysis identifies triterpenoid-induced defense responses in Myzus persicae Sülzer-infested peach.

The defense response of peach (Prunus persica) to insect attack involves changes in gene expression and metabolites. Piercing/sucking insects such as green peach aphid cause direct damage by obtaining phloem nutrients and indirect damage by spreading plant viruses. To investigate the response of peach trees to aphids, the leaf transcriptome and metabolome of two genotypes with different sensitivities to green peach aphid (GPA, Myzus persicae) were studied. The transcriptome analysis of infected peach leaves showed two different response patterns. The gene expression of aphid-susceptible peach plants infected by aphids was more similar to that of the control plants, while the gene expression of aphid-resistant peach plants infected by aphids showed strongly induced changes in gene expression compared with the response in the control plants. Furthermore, gene transcripts in defense-related pathways, including plant-pathogen interaction, MAPK signaling, and several metabolic pathways, were more strongly enriched upon aphid infestation. Untargeted secondary metabolite profiling confirmed that aphid treatment induced larger changes in aphid-resistant peaches than in aphid-susceptible peaches. Consistent with transcriptomic alterations, nine triterpenoids showed extremely significant GPA-induced accumulation in aphid-resistant peaches, whereas triterpenoid abundance remained predominantly unchanged or undetected in aphid- susceptible peaches. Furthermore, some types of transcription factors (including WRKYs, ERFs, NACs, etc.) were more strongly induced upon GPA infestation in aphid-resistant peaches but not in aphid-susceptible peaches. Aphid feeding-dependent transcriptome and metabolite profiles provide the foundation for understanding the molecular mechanisms underlying the response of peach to aphid infestation. These results suggested that accumulation of specialized triterpenoids and the corresponding pathway transcripts may play a key role in peach GPA resistance.

Impact of Varying Dietary Calcium Contents on the Gut Metabolomics of Yunnan Semi-Fine Wool Sheep (Ovis aries).

Yunnan semi-fine wool (YSFW) is a recently developed dual-purpose (meat and wool) sheep breed mainly found in Yunnan Province, China. Moreover, dietary calcium is essential for animal health and productivity. The current experiment aimed to investigate the impact of dietary calcium on sheep gut metabolite profile. For this, thirty YSFW rams (male, age = 10 months, and body weight = 40.37 ± 0.49 kg) were randomized into three groups (n = 10 rams/group), followed by a completely randomized design, and the groups were allotted to one of three dietary calcium levels (Q_1 = 0.50%, Q_3 = 0.73%, and Q_5 = 0.98% on a dry basis). The rams were fed ad libitum by feeding twice a day (at 08:00 and 17:00 h/day) throughout the experimental period (44 day). On the 21st day of the experiment, fecal samples were collected from 27 rams (9/group) and untargeted metabolite profiling was performed by using ultra-performance liquid chromatography. The PCA plot showed that the Q_5 group metabolites were clustered more tightly than for Q_1 and Q_3, respectively. The tightly clustering molecules were mainly alkaloids and their derivatives, benzenoids, lignans and related compounds, lipids, nucleotides, organic acids, and nitrogenous-based derivatives. According to the Kyoto Encyclopedia of Genes and Genomes pathway analysis, these molecules potentially contribute to metabolic pathways, biosynthesis of secondary metabolites, proteinaceous compounds, and the metabolism of the protein derivatives, particularly amino acids. The PLS-DA plots revealed a significant difference between the Q_1, Q_3, and Q_5 groups, suggesting that Q_5 had a clear separation across the groups. Based on the metabolomic analysis, feeding different levels of dietary calcium significantly changed the metabolomic profile of YSFW rams, which primarily entails metabolic pathways such as energy, protein, and lipid metabolism.

Associations of neighborhood sociodemographic environment with mortality and circulating metabolites among low-income black and white adults living in the southeastern United States

BackgroundResiding in a disadvantaged neighborhood has been linked to increased mortality. However, the impact of residential segregation and social vulnerability on cause-specific mortality is understudied. Additionally, the circulating metabolic correlates of neighborhood sociodemographic environment remain unexplored. Therefore, we examined multiple neighborhood sociodemographic metrics, i.e., neighborhood deprivation index (NDI), residential segregation index (RSI), and social vulnerability index (SVI), with all-cause and cardiovascular disease (CVD) and cancer-specific mortality and circulating metabolites in the Southern Community Cohort Study (SCCS).MethodsThe SCCS is a prospective cohort of primarily low-income adults aged 40–79, enrolled from the southeastern United States during 2002–2009. This analysis included self-reported Black/African American or non-Hispanic White participants and excluded those who died or were lost to follow-up ≤ 1 year. Untargeted metabolite profiling was performed using baseline plasma samples in a subset of SCCS participants.ResultsAmong 79,631 participants, 23,356 deaths (7214 from CVD and 5394 from cancer) were documented over a median 15-year follow-up. Higher NDI, RSI, and SVI were associated with increased all-cause, CVD, and cancer mortality, independent of standard clinical and sociodemographic risk factors and consistent between racial groups (standardized HRs among all participants were 1.07 to 1.20 in age/sex/race-adjusted model and 1.04 to 1.08 after comprehensive adjustment; all P < 0.05/3 except for cancer mortality after comprehensive adjustment). The standard risk factors explained < 40% of the variations in NDI/RSI/SVI and mediated < 70% of their associations with mortality. Among 1110 circulating metabolites measured in 1688 participants, 134 and 27 metabolites were associated with NDI and RSI (all FDR < 0.05) and mediated 61.7% and 21.2% of the NDI/RSI-mortality association, respectively. Adding those metabolites to standard risk factors increased the mediation proportion from 38.4 to 87.9% and 25.8 to 42.6% for the NDI/RSI-mortality association, respectively.ConclusionsAmong low-income Black/African American adults and non-Hispanic White adults living in the southeastern United States, a disadvantaged neighborhood sociodemographic environment was associated with increased all-cause and CVD and cancer-specific mortality beyond standard risk factors. Circulating metabolites may unveil biological pathways underlying the health effect of neighborhood sociodemographic environment. More public health efforts should be devoted to reducing neighborhood environment-related health disparities, especially for low-income individuals.

Untargeted metabolomics, optimization of microwave-assisted extraction using Box-Behnken design and evaluation of antioxidant, and antidiabetic activities of sugarcane bagasse.

The fruit wastes, in particular agricultural wastes, are considered potential and inexpensive sources of bioactive compounds. The current study was aimed at the preparation of an optimized extract of sugarcane bagasse using microwave-assisted extraction (MAE) technology and comparative evaluation of chemical composition, antioxidant, and antidiabetic activities with extract prepared through maceration technique. Box-Behnken Design (BDD) with response surface methodology was applied to observe interactions of three independent variables (ethanol concentrations [%], microwave power [W], and extraction time [min]) on the dependent variables (total phenolic content [TPC] and antioxidant status via 2,2-diphenyl-1-picrylhydrazyl [DPPH] to establish optimal extraction conditions. The ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) analysis was applied for untargeted metabolite profiling, and in vitro assays were used for evaluation of the antidiabetic and antioxidant potential of the extract. Moreover, an in silico study was used to predict the interaction of five dominant compounds from the UHPLC-Q-TOF-MS profile against the dipeptidyl peptidase-IV (DPP-IV) enzyme. The optimal conditions for the extraction were established at 60% (v/v) ethanol, 500 W microwave power, and 5 min time with TPC 12.83 ± 0.66 mg GAE/g d.w. and DPPH 45.09 ± 0.07%. The UHPLC-Q-TOF-MS analysis revealed the presence of a total of 106 compounds in the extract. Moreover, the extract prepared through MAE technology presented higher TPC and DPPH findings than the extract prepared through maceration. Similarly, the extract was also found with good antidiabetic activity by inhibiting the DPP-IV enzyme which was also rectified theoretically by a molecular docking study. The current study presents a sustainable and an optimized approach for the preparation of sugarcane bagasse extract with functional phytoconstituents and higher antidiabetic and antioxidant activities.

A metabolomics analysis of interspecies and seasonal trends in ruminant milk: The molecular difference between bovine, caprine, and ovine milk

Ruminant milk composition can be affected by many factors, primarily interspecies differences, but also environmental factors (e.g., season, feeding system, and feed composition). Pasture-based feeding systems are known to be influenced by seasonal effects on grass composition. Spring pasture is rich in protein and low in fiber compared with late-season pasture, potentially inducing variability in the composition of some milk metabolites across the season. This study aimed to investigate interspecies and seasonal differences in the milk metabolome across the 3 major commercial ruminant milk species from factories in New Zealand: bovine, caprine, and ovine milk. Samples of bovine (n = 41) and caprine (n = 44) raw milk were collected monthly for a period of 9 mo (August 2016-April 2017), and ovine milk samples (n = 20) were collected for a period of 5 mo (August 2016-January 2017). Milk samples were subjected to biphasic extraction, and untargeted metabolite profiling was performed using 2 separate liquid chromatography high-resolution mass spectrometry analytical methods (polar metabolites and lipids). Major differences in the milk metabolome were observed between the 3 ruminant species, with 414 of 587 (71%) polar metabolite features and 210 of 233 (87%) lipid features being significantly different between species. Significant seasonal trends were observed in the polar metabolite fraction for bovine, caprine, and ovine milk (17, 24, and 32 metabolites, respectively), suggesting that the polar metabolite relative intensities of ovine and caprine milk were more susceptible to changes within seasons than bovine milk. We found no significant seasonal difference for the triglycerides (TG) species measured in bovine milk, whereas 3 and 52 TG species changed in caprine and ovine milk, respectively, across the seasons. In addition, 4 phosphatidylcholines and 2 phosphatidylethanolamines varied in caprine milk within the season, and 8 diglycerides varied in ovine milk. The interspecies and seasonal metabolite differences reported here provide a knowledge base of components potentially linked to milk physiochemical properties, and potential health benefits of New Zealand pasture-fed dairy ingredients.

A traditional fermented bamboo shoot reduces intracellular fat accumulation and promotes fat browning in differentiated 3T3‐L1 adipocyte cells through the activation of the AMPK signaling pathway

AbstractFunctional foods, such as fermented bamboo shoots, have a long history of consumption among the ethnic communities in northeast India. These locally fermented bamboo shoots contain a wealth of beneficial microbes and metabolites that can help combat metabolic syndromes like obesity. However, the precise effects and mechanism behind fermented bamboo shoot products and their anti‐obesity properties remain unknown. This study aims to explore the different types of fermented bamboo shoot products to determine their potential anti‐obesity effects as well as to analyze their microbial diversity and metabolite profiles. Using both culture‐dependent and culture‐independent methods, we found a high abundance of lactic acid bacteria from the Firmicutes and Proteobacteria phyla in the sample. Gas chromatography–mass spectrometry (GC–MS) based untargeted metabolite profiling detected several aroma‐active compounds, bioactive metabolites, short‐chain fatty acids, and essential amino acids in the samples. The water extract derived from a particular type of fermented bamboo shoot, Melye‐amiley, was found to significantly reduce intracellular lipid accumulation in cultured 3T3‐L1 cells. In addition, this extract increased the expression of lipolytic (hormone‐sensitive lipase, lipoprotein lipase, and adipose triglyceride lipase) and browning regulator genes (uncoupling protein [UCP1], PRDM16, and PGC1‐alpha). By activating the AMPK signaling pathway, the water extract from Melye‐amiley also upregulated thermogenic protein expression and promoted mitochondrial biogenesis and fatty acid β‐oxidation. These findings suggest that fermented bamboo shoot extract has promising anti‐obesity effects by boosting energy expenditure in white adipocytes. Future research is necessary to identify the active ingredient(s) that may lead to new therapies to treat obesity.

Comprehensive mass spectrometric metabolomic profiling of a chemically diverse collection of plants of the Celastraceae family

Natural products exhibit interesting structural features and significant biological activities. The discovery of new bioactive molecules is a complex process that requires high-quality metabolite profiling data to properly target the isolation of compounds of interest and enable their complete structural characterization. The same metabolite profiling data can also be used to better understand chemotaxonomic links between species. This Data Descriptor details a dataset resulting from the untargeted liquid chromatography-mass spectrometry metabolite profiling of 76 natural extracts of the Celastraceae family. The spectral annotation results and related chemical and taxonomic metadata are shared, along with proposed examples of data reuse. This data can be further studied by researchers exploring the chemical diversity of natural products. This can serve as a reference sample set for deep metabolome investigation of this chemically rich plant family.

Metabolic diversity, biosynthetic pathways, and metabolite biomarkers analysed via untargeted metabolomics and the antioxidant potential reveal for high temperature tolerance in tomato hybrid

High-temperature tolerant varieties/hybrids of tomato overcome stress-related changes due to their intrinsic cellular metabolic capabilities. We have used untargeted metabolite profiling and comparative biochemical and biological analyses of high temperature tolerant (HTT) tomato hybrid VRNTH18283 and sensitive (HTS) VRNTH19072 to decipher altered metabolic pathways and biomarker metabolites which were linked with enhanced antioxidant activity in HTT fruits. Parameters like fruit weight, yield per plant and key biochemical determinants viz. total soluble sugar (TSS), titratable acidity, ascorbic acid and lycopene content were significantly high in HTT hybrid compared to HTS. Metabolomics revealed upregulated metabolite diversity in HTT fruit extract (11,453 m/z features) as compared to 8834 m/z features in HTS. Statistical analysis revealed 423 differentially up-regulated and 410 down-regulated metabolite features. Multivariate data analyses showed sample discrimination based on distinct phytochemical diversification. Functionally annotated metabolite features showed enrichment in metabolic pathways including linoleic and linolenic acid metabolism, monoterpenoids biosynthesis and degradation, cutin, suberin and wax biosynthesis, and sphingolipids metabolism. Metabolite features were categorized in benzamides, amino acids, sugars, nucleotides, sterols, glycosides, phenylacetaldehydes, indoles, tryptamines and organic acids as major compound classes. Among 22 potentially discriminatory metabolites obtained from OPLS-DA model, all including prominently abundant solasodine, thiamine diphosphate, L-tryptophan, folic acid, lycopene, dihydrozeatin, myricetin, tomatidine, chlorogenic acid and α-tocotrienol were classified as metabolite biomarkers and were correlated with enhanced antioxidant activity. The study revealed that the altered physicochemical profile, increased metabolite compositional diversity and discriminatory metabolite biomarkers are liable for improved performance of the hybrid HTT VRNTH18283 against the negative impact of high temperature in comparison to HTS VRNTH19072.

Metabolites Profile of Extracts and Fractions of Erythroxylum mexicanum Kunth by UHPLC-QTOF-MS/MS and its Antibacterial, Cytotoxic and Nitric Oxide Inhibitory Activities.

The present study shows the untargeted metabolite profiling and in vitro antibacterial, cytotoxic, and nitric oxide (NO) inhibitory activities of the methanolic leaves extract (MLE) and methanolic stem extract (MSE) of Erythroxylum mexicanum, as well as the fractions from MSE. Using ultra-high performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS/MS), a total of 70 metabolites were identified; mainly alkaloids in the MLE, while the MSE showed a high abundance of diterpenoids. The MSE fractions exhibited differential activity against Gram-positive bacteria. Notably, the hexane fraction (HSF) against Streptococcus pyogenes ATCC 19615 (MIC=62.5 μg/mL) exhibited a bactericidal effect. The MSE fractions exhibited cytotoxicity against all cancer cell lines tested, with selectivity towards them compared to a noncancerous cell line. Particularly, the HSF and chloroform fraction (CSF) showed the highest cytotoxicity against prostate cancer (PC-3) cells, with IC50 values of 19.9 and 18.1 μg/mL and selectivity indexes of 3.8 and 4.2, respectively. Both the HSF and ethyl acetate (EASF) fractions of the MSE inhibited NO production in RAW 264.7 macrophages, with NO production percentages of 50.0 % and 51.7 %, respectively, at a concentration of 30 μg/mL. These results indicated that E. mexicanum can be a source of antibacterial, cytotoxic, and anti-inflammatory metabolites.

Dietary patterns, untargeted metabolite profiles and their association with colorectal cancer risk

We investigated data-driven and hypothesis-driven dietary patterns and their association to plasma metabolite profiles and subsequent colorectal cancer (CRC) risk in 680 CRC cases and individually matched controls. Dietary patterns were identified from combined exploratory/confirmatory factor analysis. We assessed association to LC–MS metabolic profiles by random forest regression and to CRC risk by multivariable conditional logistic regression. Principal component analysis was used on metabolite features selected to reflect dietary exposures. Component scores were associated to CRC risk and dietary exposures using partial Spearman correlation. We identified 12 data-driven dietary patterns, of which a breakfast food pattern showed an inverse association with CRC risk (OR per standard deviation increase 0.89, 95% CI 0.80–1.00, p = 0.04). This pattern was also inversely associated with risk of distal colon cancer (0.75, 0.61–0.96, p = 0.01) and was more pronounced in women (0.69, 0.49–0.96, p = 0.03). Associations between meat, fast-food, fruit soup/rice patterns and CRC risk were modified by tumor location in women. Alcohol as well as fruit and vegetables associated with metabolite profiles (Q2 0.22 and 0.26, respectively). One metabolite reflecting alcohol intake associated with increased CRC risk, whereas three metabolites reflecting fiber, wholegrain, and fruit and vegetables associated with decreased CRC risk.

Anticancer and anti-inflammatory activity and untargeted metabolite profiling by UPLC-ESI-QTOF of crude extracts of mangrove endophytic fungus Aspergillus stellatus LM-03

Mangrove ecosystem is diverse habitat for number of medicinally important microorganisms including fungi. Scientific research from last three decade emphasises it potential in important secondary metabolites production, which have wide biological activities. The current study elaborates isolation of fungi from pneumatophore of mangrove plant Avicennia marina and investigation of ethyl acetate extract for antimicrobial, anti-inflammatory and anticancer activity. Ethyl acetate extract of the fungus displayed maximum anti-inflammatory activity with 76.39% inhibition for by protein denaturation method and 22.52% inhibition for membrane stabilisation method with respect to standard ibuprofen. Further, the anticancer activity of Aspergillus stellatus LM-03 exhibited against MCF-7 of breast cancer cell with an IC50 value of 33.24 µg/ml. HPLC and UPLC-ESI-QTOF analysis displayed nine compound mainly alternariol monomethyl ether and methyl jasmonate, which are highly anticancer activity and moderate anti-inflammatory activity with other compounds as angelic anhydride, DL-3-(4-Hydroxyphenyl)lactic acid, 8,9-epoxyl-3-isobutyryloxy-10-(2-methylbutanoyl)thymol, 5-(6-methyl-7-oxooctyl) furan-2(5H)-one and phenyl-butyryl-glutamine with an unidentified compounds. The results of the current study indicate that it may be worthwhile to investigate Aspergillus stellatus LM-03 extract for promising bioactive metabolites with potential for medicinal use.

Multi-omics analysis of hospital-acquired diarrhoeal patients reveals biomarkers of enterococcal proliferation and Clostridioides difficile infection

Hospital-acquired diarrhoea (HAD) is common, and often associated with gut microbiota and metabolome dysbiosis following antibiotic administration. Clostridioides difficile is the most significant antibiotic-associated diarrhoeal (AAD) pathogen, but less is known about the microbiota and metabolome associated with AAD and C. difficile infection (CDI) with contrasting antibiotic treatment. We characterised faecal microbiota and metabolome for 169 HAD patients (33 with CDI and 133 non-CDI) to determine dysbiosis biomarkers and gain insights into metabolic strategies C. difficile might use for gut colonisation. The specimen microbial community was analysed using 16 S rRNA gene amplicon sequencing, coupled with untargeted metabolite profiling using gas chromatography-mass spectrometry (GC-MS), and short-chain fatty acid (SCFA) profiling using GC-MS. AAD and CDI patients were associated with a spectrum of dysbiosis reflecting non-antibiotic, short-term, and extended-antibiotic treatment. Notably, extended antibiotic treatment was associated with enterococcal proliferation (mostly vancomycin-resistant Enterococcus faecium) coupled with putative biomarkers of enterococcal tyrosine decarboxylation. We also uncovered unrecognised metabolome dynamics associated with concomitant enterococcal proliferation and CDI, including biomarkers of Stickland fermentation and amino acid competition that could distinguish CDI from non–CDI patients. Here we show, candidate metabolic biomarkers for diagnostic development with possible implications for CDI and vancomycin-resistant enterococci (VRE) treatment.

Automated Composition Assessment of Natural Extracts: Untargeted Mass Spectrometry-Based Metabolite Profiling Integrating Semiquantitative Detection.

Recent developments in mass spectrometry-based metabolite profiling allow unprecedented qualitative coverage of complex biological extract composition. However, the electrospray ionization used in metabolite profiling generates multiple artifactual signals for a single analyte. This leads to thousands of signals per analysis without satisfactory means of filtering those corresponding to abundant constituents. Generic approaches are therefore needed for the qualitative and quantitative annotation of a broad range of relevant constituents. For this, we used an analytical platform combining liquid chromatography-mass spectrometry (LC-MS) with Charged Aerosol Detection (CAD). We established a generic metabolite profiling for the concomitant recording of qualitative MS data and semiquantitative CAD profiles. The MS features (recorded in high-resolution tandem MS) are grouped and annotated using state-of-the-art tools. To efficiently attribute features to their corresponding extracted and integrated CAD peaks, a custom signal pretreatment and peak-shape comparison workflow is built. This strategy allows us to automatically contextualize features at both major and minor metabolome levels, together with a detailed reporting of their annotation including relevant orthogonal information (taxonomy, retention time). Signals not attributed to CAD peaks are considered minor metabolites. Results are illustrated on an ethanolic extract of Swertia chirayita (Roxb.) H. Karst., a bitter plant of industrial interest, exhibiting the typical complexity of plant extracts as a proof of concept. This generic qualitative and quantitative approach paves the way to automatically assess the composition of single natural extracts of interest or broader collections, thus facilitating new ingredient registrations or natural-extracts-based drug discovery campaigns.

Biomarker Development for Identifying Mud Loach (Misgurnus mizolepis) Origin Country Using Untargeted Metabolite Profiling.

Mud loach (Misgurnus mizolepis) has long been consumed in Korea. Recently, Chinese mud loaches were replaced with expensive Korean mud loaches, owing to taste and preference. Such issues occur in aquatic food distribution processes, leading to inferior food delivery. Previously, a study was conducted to confirm the origin of mud loaches using genetic analysis. However, untargeted metabolites profiling of mud loaches has not been reported. Untargeted metabolomics provides information on the overall metabolic profiling of a sample, allowing the identification of new metabolites. Here, we analyzed the metabolites of mud loaches of different geographical origins using liquid chromatography (LC)-quadrupole-time-of-flight mass spectrometry (MS). Orthogonal partial least squares discriminant analysis from LC/MS datasets showed a clear distinction between Korean and Chinese mud loaches, and univariate statistical analysis showed significantly different metabolites between them. N-acetylhistidine and anserine were selected as biomarkers for geographical origin discrimination using the receiver operating characteristic curve. N-acetylhistidine and anserine levels were significantly higher in Chinese than in Korean mud loaches. These results indicate that metabolic analysis can be used to discriminate between the geographical origins of mud loaches, curtailing the inadvertent substitution of mud loaches from different regions.

Physiological and biochemical traits positively modulate tissue-specific withanolides and untargeted metabolites in Withania somnifera (L.) dunal under salinity stress

Withania somnifera (L.) Dunal (Ashwagandha) has been used in herbal medicines worldwide and in the Indian traditional medicinal system for 3000 years. It is a member of the Solanaceae family distributed across Asia, Africa, Australia, and Europe. Its bioactive secondary metabolite (withanolide) biosynthesis is sensitive to salinity stress, though the mechanism remains unexplored. Therefore, we investigated the effect of Sodium chloride (NaCl) on growth, photosynthesis, biochemical traits, tissue-specific withanolide, and untargeted metabolites in W. somnifera. Ashwagandha plants were raised in pots containing soil mixture and treated with different NaCl concentrations (0 as control, 10, 30, and 50 mM) for one month inside the greenhouse. NaCl stress significantly enhanced withaferin A (WFA) (3.79 mg/g), withanolide A (WA) (0.51 mg/g), and withanone (WN) (0.022 mg/g) at 50 mM NaCl groups in the shoot. Similarly, in the root, a significant increase in WFA (0.19 mg/g) and WN (0.0016 mg/g) were observed at 10 mM, WA (0.059 mg/g) at 30 mM, and withanolide B (WB) (0.013 mg/g) at 50 mM NaCl groups compared to control. LC-MS-based untargeted metabolite profiling revealed 37 differentially accumulated metabolites in all groups. Maximum abundance of glycyl-hydroxyproline (8X) followed by tyrosyl-valine (2X) and 3-hydroxy-beta-ionone (2X) were recorded at 50 mM NaCl groups compared to the control. This study showed for the first time that low NaCl stress enhances the biosynthesis of tissue-specific withanolides through physio-biochemical and metabolites adjustment. Overall, we demonstrated a multifaceted approach for cultivating medicinal crops in salt-affected areas with enhanced bioactive metabolites for healthcare and pharmaceutical industries.

Preliminary Phytochemical and Biological Evaluation of Rudbeckia hirta Flowers.

Black-eyed Susan (Rudbeckia hirta L.), a flowering plant with various traditional medicinal uses, has recently garnered interest for its therapeutic properties. However, little is known about the potential therapeutic activities of the plant species. The current study focused on conducting a comprehensive investigation into the chemical composition and bioactivity of black-eyed Susan cultivated in Romania. Untargeted metabolite profiling and UHPLC-HR-MS phytochemical analysis of the studied extract revealed the presence of more than 250 compounds pertaining to different classes, including sesquiterpene lactones, polyphenolic acids, flavonoids, amino acids, and fatty acids. The tested extract exhibited inhibitory activity against Gram-positive bacteria and showed promising antifungal activity. It also demonstrated potent antioxidant properties through iron chelation and 15-LOX inhibition capacities, as well as inhibition of cell growth, particularly on the MCF-7 cell line, suggesting potential anticancer effects. Therefore, current research provides valuable information on the antioxidant, antimicrobial, and antitumor potential of Rudbeckia hirta flowers. Implicitly, the discovery of such a wide range of biosubstances, together with the biological activity observed for the studied extract in these preliminary in vitro studies, paves the way for future investigation of the potential application of the plant in the pharmaceutical and nutraceutical sectors.

Comparative Analysis of the Physicochemical Properties and Metabolites of Farinose and Crisp Lotus Roots (Nelumbo nucifera Gaertn.) with Different Geographical Origins.

Lotus roots are widely consumed vegetables because of their great taste and abundant nutrients, but their quality varies with the environments and cultivar. This study systematically compared farinose (Elian No. 5) and crisp (Elian No. 6) lotus root cultivars from three geographical origins. Pasting and texture characteristics verified that Elian No. 5 possessed lower hardness and lower ability to withstand shear stress and heating during cooking compared with Elian No. 6. Untargeted metabolite profiling was first performed using ultrahigh-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) combined with a Zeno trap. In total, 188 metabolites were identified based on the matching chemistry database. Multivariate analysis demonstrated that lotus roots from different cultivars and origins could be adequately distinguished. Sixty-one differential metabolites were identified among three Elian No. 5 samples, and 28 were identified among three Elian No. 6 samples. Isoscopoletin, scopoletin, and paprazine were the most differential metabolites between Elian No. 5 and Elian No. 6. These results can inform future research on the discrimination and utilization of lotus roots.

Metabolite profiling identifies chemical markers associated with the cytotoxic properties of roasted fermented avocado seeds

Studies have demonstrated avocado seeds are a good source of bioactive compounds. This study investigated the effects of roasting on the metabolites and anticancer activities of fermented avocado seeds. All three anti-cancer activities of fermented avocado seeds were higher at lower roasting temperature and time. The best inhibition effect was found against Hep G2 followed by the MDA-MB-231 and MCF-7 cancer cell lines. Untargeted metabolite profiling using gas chromatography-mass spectrometry resulted in identification of 208 metabolites. In total, 41 metabolites identified had VIP values more than 1 using PLS-R that were related to anticancer activities. All amino acids and most sugars were higher at lower roasting temperature and positively correlated to anticancer activity. The roasting conditions for optimal antioxidant and anticancer activities were determined to be 121 °C for 9 min. Findings showed that fermented avocado seed powder has the potential to become a functional food ingredient with beneficial bioctive properties.