Using Ultra-high-performance Liquid Chromatography-mass Spectrometry Research Articles

The Correlation between Chemical Ingredients and Acute Toxicity of Psoraleae Fructus and Two Classic Prescriptions

Objective: To compare the acute toxicity and chemical ingredients of Psoraleae Fructus (PF) with those of two classic prescriptions, Ershen Wan (ESW) and Sishen Wan (SSW). Methods: Based on classical toxicological methods, body weight, food and water consumption, lethal conditions, and toxic reactions were recorded after administering single oral doses of PF, ESW, and SSW. The 50% lethal dose (LD50) values of PF and ESW and the maximum tolerance dose (MTD) of SSW were determined. In addition, PF, ESW, and SSW constituents were detected using ultra-high-performance liquid chromatography/mass spectrometry (UPLC-MS), and the spectrum-toxicity correlation was analyzed. Results: The LD50 of PF and ESW were 53.9 g/kg/d (46.2–63.0 g/kg/d, 95% CL) and 68.3 g/kg/d (59.0–78.9 g/kg/d, 95% CL), which were respectively about 40 times and 50 times of the human daily dosage. The MTD of SSW was 41.0 g/kg/d, indicating the highest safety. The chemical ingredients and toxicity correlation analyses inferred that compatibility reduced the contents of 13 potential hepatotoxin compounds in PF. Conclusions: The classic compatibility of ESW and SSW effectively attenuated the hepatotoxicity of PF, which was related to the reduced content of potentially toxic substances, particularly coumarins. This study explored the principles of attenuating the toxicity of classic prescriptions to provide a reference for the rational clinical use of PF.

Serum metabolomics analysis combined with network pharmacology reveals possible mechanisms of postoperative cognitive dysfunction in the treatment of Mongolian medicine Eerdun Wurile basic formula.

This study analyzed the endogenous metabolites and metabolic pathways in the serum of Sprague-Dawley (SD) rats gavaged with the Eerdun Wurile basic formula (EWB) using metabolomics methods and network pharmacology to explore the possible mechanism of action of the EWB in improving postoperative cognitive dysfunction (POCD). SD rats were divided into the basic formula group, which received the EWB, and the control group, which received equal amounts of distilled water. The blood was collected from the abdominal aorta and analyzed for metabolite profiles using ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS). Network pharmacology predicts the targets of the differential metabolites and disease targets; takes the intersection and constructs a "metabolite-disease-target" network; and performs protein-protein interaction, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes analyses. A total of 56 metabolites were selected for significant differences between the groups, mainly affecting amphetamine addiction, alcoholism, and regulation of lipolysis in adipocytes. A total of 177 potential targets for differential metabolite action in POCD were selected. The PI3K-Akt pathway, the HIF-1 pathway, and the FoxO pathway were in key positions. The studies have shown that EWB could improve POCD through multicomponents, multitargets, and multipathways, providing new possibilities and reference values for the treatment of POCD.

Antioxidant, Anti-Diabetic, and Anti-Inflammation Activity of Garcinia livingstonei Aqueous Leaf Extract: A Preliminary Study.

Diabetes mellitus (DM) is the second leading cause of mortality globally. The increased concern for DM is due to the underlying complications accompanying hyperglycaemia, associated with oxidative stress and consequent inflammation. The investigation of safe and effective treatments for DM is necessary. In the present study, the cytotoxicity, phytochemical analysis, antioxidant capacity, anti-inflammatory, and antidiabetic effects in an aqueous extract of Garcinia livingstonei leaves were assessed. All tested extract concentrations showed no toxicity against C3A hepatocytes. Several phenolic compounds were identified using ultra-high performance liquid chromatography mass spectrometry (UHPLC-MS). The total polyphenol content was 100.9741 mg GAE/g, 16.7712 mg CE/g flavanols, and 2.3548 mg QE/g flavonols. The antioxidant capacity values were 253.4268 mg AAE/g, 192.232 mg TE/g, and 167.8724 mg TE/g for ferric reducing antioxidant power (FRAP), Trolox equivalent antioxidant capacity (TEAC), and 2,2-diphenyl-1-pycrylhydrazyl (DPPH), respectively. The plant extract significantly (p < 0.05) demonstrated anti-inflammatory and hypoglycaemic effects in a dose-dependent manner, with the α-glucosidase inhibition of the extract being higher (p < 0.05) than in the standard conventional drug (acarbose). The findings of this study revealed the potential of the constituents of G. livingstonei aqueous leaf extract in DM treatment. Further studies on the preparation and mechanisms of action of the plant in DM treatment are recommended.

Isolation and identification of forced degradation products of Febuxostat

AbstractThe current study explains the degradation behavior of Febuxostat API, a non‐purine xanthine oxidase inhibitor used to treat hyperuricemia. A degradation study was carried out as per the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines, and the study confirms that the Febuxostat is largely stable in thermal, photolytic, oxidative, and basic hydrolytic conditions and labile in acid hydrolysis conditions. There were four different degradation products (DPs) found during acid hydrolysis; of these, DPs 2, 3, and 4 are new and have never been reported before, while DP 1 is known and has already been published. All these DPs were identified using ultra‐high‐performance liquid chromatography‐mass spectrometry (UHPLC‐MS) analysis, purified by using preparative HPLC, and characterized using high‐resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) spectroscopy techniques. The formed DPs are by the hydrolysis of the cyano functional group of Febuxostat and the esterification of DP‐1 under acidic conditions. All DP's structural characterization was carried out using NMR spectroscopy and HRMS. The present study describes concrete confirmation of DP structures and it explains the stability behavior of the Febuxostat. The current method is also used to identify DPs with shorter runtime in the future.

Metabolomic analysis of the human placenta reveals perturbations in amino acids, purine metabolites, and small organic acids in spontaneous preterm birth.

Spontaneous preterm delivery presents one of the most complex challenges in obstetrics and is a leading cause of perinatal morbidity and mortality. Although it is a common endpoint for multiple pathological processes, the mechanisms governing the etiological complexity of spontaneous preterm birth and the placental responses are poorly understood. This study examined placental tissues collected between May 2019 and May 2022 from a well-defined cohort of women who experienced spontaneous preterm birth (n = 72) and healthy full-term deliveries (n = 30). Placental metabolomic profiling of polar metabolites was performed using Ultra-High Performance Liquid Chromatography/Mass Spectrometry (UHPLC/MS) analysis. The resulting data were analyzed using multi- and univariate statistical methods followed by unsupervised clustering. A comprehensive metabolomic evaluation of the placenta revealed that spontaneous preterm birth was associated with significant changes in the levels of 34 polar metabolites involved in intracellular energy metabolism and biochemical activity, including amino acids, purine metabolites, and small organic acids. We found that neither the preterm delivery phenotype nor the inflammatory response explain the reported differential placental metabolome. However, unsupervised clustering revealed two molecular subtypes of placentas from spontaneous preterm pregnancies exhibiting differential enrichment of clinical parameters. We also identified differences between early and late preterm samples, suggesting distinct placental functions in early spontaneous preterm delivery. Altogether, we present evidence that spontaneous preterm birth is associated with significant changes in the level of placental polar metabolites. Dysregulation of the placental metabolome may underpin important (patho)physiological mechanisms involved in preterm birth etiology and long-term neonatal outcomes.

Windows Scanning Multiomics: Integrated Metabolomics and Proteomics.

Metabolomics and proteomics offer significant advantages in understanding biological mechanisms at two hierarchical levels. However, conventional single omics analysis faces challenges due to the high demand for specimens and the complexity of intrinsic associations. To obtain comprehensive and accurate system biological information, we developed a multiomics analytical method called Windows Scanning Multiomics (WSM). In this method, we performed simultaneous extraction of metabolites and proteins from the same sample, resulting in a 10% increase in the coverage of the identified biomolecules. Both metabolomics and proteomics analyses were conducted by using ultrahigh-performance liquid chromatography mass spectrometry (UPLC-MS), eliminating the need for instrument conversions. Additionally, we designed an R-based program (WSM.R) to integrate mathematical and biological correlations between metabolites and proteins into a correlation network. The network created from simultaneously extracted biomolecules was more focused and comprehensive compared to those from separate extractions. Notably, we excluded six pairs of false-positive relationships between metabolites and proteins in the network established using simultaneously extracted biomolecules. In conclusion, this study introduces a novel approach for multiomics analysis and data processing that greatly aids in bioinformation mining from multiomics results. This method is poised to play an indispensable role in systems biology research.

Analysis of total thiol concentrations in environmental samples using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS)

In this study, we developed an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) approach to measure the concentration of total reactive thiol binding sites in a range of types of environmental samples by monitoring the decrease of monobromo-(trimethylammonio)-bimane (qBBr) caused by thiol-qBBr binding reactions. We first examined the stability of qBBr and potential side reactions that could reduce the accuracy of the UHPLC-MS approach. We found that significant loss of qBBr occurs in the absence of thiols in aqueous solutions after 2–4 h due to qBBr degradation and other side reactions, such as a chloride-for-bromide substitution reaction. To address these issues, in our approach we conduct the thiol-qBBr binding reaction in the presence of excessive qBBr for only 1 h, and we compare the MS signal of unbound qBBr in a thiol-free qBBr standard with that in a thiol-bearing qBBr standard in order to calculate the decrease in the concentration of free qBBr in solution that is caused by thiol-qBBr binding. Using 100–600 nM of N-Acetyl-L-cysteine (ACYS) as a model thiol in a 2 μM qBBr standard, we found that the measured decrease in the MS signal of unbound qBBr linearly correlates to the concentration of added ACYS, with a R2 of 0.988, demonstrating that monitoring the decrease in unbound qBBr can be used to calculate the concentration of total thiol sites in a sample. We demonstrate that the new UHPLC-MS approach yields accurate results when measuring a series of single and mixed thiol standards in the 200–500 nM range, with measured values ranging from 86% to 109% of the known concentration of thiols. In addition to the above control tests, we also used the UHPLC-MS approach to measure dissolved thiol concentrations in several lake water samples. These measurements revealed that each lake contained low μM levels of total thiols, not only demonstrating the ability of the analytical approach to determine thiol site concentrations in natural waters, but also suggesting the widespread importance of these sites in controlling chalcophile element behavior in these systems. The UHPLC-MS approach not only can be used to determine the concentration of dissolved thiol sites at nM levels, but can also be adapted to measure the concentration of thiol binding sites on other environmental samples such as soil, sediment, and solid-phase organic matter surfaces.

Membrane phospholipids and nucleotide derivatives in “big six” Escherichia coli are targets of chlorine-based sanitisers during lettuce decontamination

Sodium chlorite (NaClO), the most commonly used sanitiser for fresh produce decontamination, is associated with the problems of pungency and corrosiveness. In search for a more environmentally- and user-friendly chlorine-based sanitiser, acidic electrolysed water (AEW), came into view in recent years. To determine whether AEW is an ideal alternative to NaClO, using ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS), this study thoroughly investigated the effectiveness of AEW and NaClO, both containing 4 mg/L of free available chlorine (FAC), in inhibiting the “big six” Escherichia coli pathogens (serotypes O26, O45, O103, O111, O121 and O145) on lettuce as well as the underlying antimicrobial mechanisms.The two sanitisers led to generally comparable E. coli cell reductions after a 10-min treatment, which indicated the decisive role of FAC in their antimicrobial effects. Among the strains, mortalities varied from approximately 0.68 log CFU/g in O111 under both treatments to 1.48 ± 0.04 log CFU/g in AEW-treated O26 and 1.23 ± 0.04 log CFU/g in NaClO-treated O45. O111's high resistance was confirmed by its almost undisturbed metabolome whereas the high lethality of O26 and O45 could be attributed to the loss of membrane phospholipids. Besides, retarded cell growth and energy deficiency as implied by a vast depletion of nucleotide derivatives further challenged O45's survival. Overall, the comparable sanitising efficacy and the similarity in antimicrobial mechanisms between the two sanitisers provided a scientific basis for replacing NaClO with the “greener” AEW for fresh produce sanitisation in the future.

Ameliorative effect of traditional polyherbal formulation on TNF-α, IL-1β and Caspase-3 expression in kidneys of wistar rats against sodium fluoride induced oxidative stress

Ethnopharmacological relevanceSharbat-e-bazoori Motadil (SBM) is a polyherbal formulation that have been used for centuries as a part of the Unani system of medicine for renal disease. Aim of the studyThe objective of this study was to explore and validate the nephroprotective potential of sugar-free SBM (SF-SBM) and its mechanisms of action against sodium fluoride (NaF)-induced nephrotoxicity in HEK-293 cells. Additionally, the study aimed to assess the quality control of SF-SBM and investigate its effects using an in vivo rat model with pattern recognition following oral administration of SF-SBM. Materials and methodsThe nephroprotective effect of SF-SBM was investigated using both an HEK-293 cell line and Wistar rats. Nephrotoxicity was induced in these models by administering NaF at a concentration of 600 ppm (parts per million) for a duration of seven days. The SF-SBM formulation was standardized using high-performance thin-layer chromatography (HPTLC) to assess the presence of marker compounds, namely gallic acid, quercetin, and ferulic acid. Metabolite characterization of SF-SBM was carried out using ultra-high-performance liquid chromatography mass spectrometry (UPLC-MS) with a monolithic capillary silica-based C18 column. This analytical technique allowed for the identification of bioactive substances and verification of the identified markers. Acute toxicity of SF-SBM was evaluated in Wistar rats by administering a single oral dose of 2000 mg/kg of SF-SBM. The nephroprotective efficacy of SF-SBM was further assessed at low (LD), medium (MD) and high (HD) doses of 32.1, 64.2, and 128.4 mg/kg, respectively, administered orally. Nephrotoxicity was induced in Wistar rats by adding NaF to their drinking water for seven days. Biochemical and urine markers were analyzed to evaluate the antioxidant, inflammatory, and apoptotic potential of SF-SBM. Additionally, histopathological analysis and immunohistochemical alterations in the expression of caspase-3 and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-4 (NOX-4) in kidney tissue were performed to confirm the findings of the in vivo experiments. Furthermore, in vivo pattern recognition of SF-SBM metabolites, identified through GC-MS metabolomics, and in-silico docking analysis of major metabolites in plasma were conducted to gain further insights. ResultPhytochemical analysis using HPTLC, TLC-bioautography, and UPLC-MS revealed the presence of several bioactive constituents in SF-SBM, including ferulic acid, gallic acid (GA), ellagic acid, quercetin, and apigenin. These compounds exhibit diverse pharmacological properties. In vitro studies demonstrated the protective effect of SF-SBM on HEK-293 cell line against nephrotoxicity. The acute toxicity study of SF-SBM at a dose of 2000 mg/kg showed no mortality or signs of toxicity throughout the 14-day observation period. In the in vivo studies, administration of NaF resulted in significant elevation (P < 0.001) of biochemical and urine parameters, indicating oxidative, inflammatory, and apoptotic stress. Histopathological examination revealed severe depletion of Bowman's capsule, and immunohistochemistry demonstrated negative immunostaining for caspase-3 and reduced NOX-4 reactions. Pre-treatment with SF-SBM significantly attenuated the elevated biochemical and urine markers, restored the antioxidant enzyme levels (such as SOD, CAT, GSH, GPx and NO), and regulated the expression of inflammatory cytokines (TNF-α, IL-1β, CASP-3) in kidney tissue at doses of SF-SBM-MD (64.2 mg/kg) and SF-SBM-HD (128.4 mg/kg), showing comparable results to those of α-Ketoanalogue. Histopathological assessment demonstrated improvements in tissue damage. Pattern recognition analysis of SF-SBM identified the presence of 56 metabolites at different time intervals. Additionally, in-silico studies revealed strong interactions of SF-SBM with a binding energy of −6.5 and −5.6 kcal for 4C2N. ConclusionThe phytoconstituents present in SF-SBM play a crucial role in its nephroprotective action by acting as potent antioxidants and reducing proinflammatory and apoptotic damage in rat cells. This indicates that SF-SBM has promising potential for the treatment of nephrotoxicity.

Metabolomics-based Approach to Analyze the Therapeutic Targets and Metabolites of a Synovitis Ointment for Knee Osteoarthritis

Background: Knee osteoarthritis (KOA) is a clinically common degenerative joint disease that is not fatal but has a high prevalence. Synovitis ointment (HMYG) is a traditional Chinese medicine formula that has been clinically proven to treat KOA; however, its therapeutic targets remain unknown. Objective: This study aimed to identify metabolites and potential targets of synovitis ointment alleviation in rats with KOA using ultra-high-performance liquid-chromatography–mass spectrometry (UHPLC-MS) metabolomics. Methods: The meniscus on each side of the knee was removed to model KOA in rats. The synovitis ointment treatment was provided for 4 weeks. The lateral diameter of the knee was measured once a week, and after 4 weeks, serum was collected to observe changes in the knee through a metabolomic analysis. Results: Synovitis ointment reduced the lateral diameter of the knee joint, relieved knee swelling, and improved knee volume. In total, 28 differential metabolites, which were mainly involved in arginine and proline metabolism and apoptosis, were identified in the Con and HMYG groups. 15-Deoxy-d-12, 14-PGJ2 and fomepizole were found to be the key metabolites after the HMYG treatment of KOA. Compared with known drugs (diclofenac diethylamine emulsion and Jin Huang San), 2-(SGlutathionyl) acetyl glutathione, daidzein, pelargonic acid, and sulfamethoxazole increased in the HMYG, and the metabolic pathways included the oxytocin signaling pathway, platelet activation, olfactory transduction, phototransduction, and cGMP-PKG signaling pathway. The expression levels of cleaved-caspase-3, Bcl-2, PIK3a, TP53, TGFB1, and NFKB1 were reversed after HMYG treatment. Conclusion: It has been observed that synovitis ointment relieves KOA. UHPLC-MS can analyze the potential mechanism of action of the herbal compound of the synovitis ointment.

Differences in Chemical Constituents between Dalbergia oliveri Heartwood and Sapwood and Their Effect on Wood Color.

The purpose of this study was to characterize and quantify the chemical constituents of heartwood and sapwood of Dalbergia oliveri extract in order to investigate the chemical components that determine the formation of heartwood's color. In this work, the types of pigments in heartwood and sapwood extract were analyzed using UV-Visible (UV) Spectrophotometer, and the main pigment components of heartwood and sapwood extract were identified and quantified using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS). The results showed that the difference in content of the main components between heartwood and sapwood of Dalbergia oliveri was slight, and the lignin structure between heartwood and sapwood is basically identical; flavonoid pigments were found to be the primary chromophoric components of heartwood and sapwood extract. However, a total of 21 flavonoids were identified in heartwood and sapwood, of which the unique substances to heartwood were vitexin, isorhamnetin, and pelargonidin, and the content of isoliquiritigenin, formononetin, and biochanin A were 253, 37, and 583 times higher in the heartwood than in the sapwood, respectively, which could be the main pigment components affecting the significant color difference between heartwood and sapwood of Dalbergia oliveri. These results will provide a foundation for revealing the underlying mechanism of color difference between heartwood and sapwood and provide a theoretical basis for wood coloring.

Metabolite Profiling and Nephroprotective Potential of the Zea mays L. Silk Extract against Diclofenac-Induced Nephrotoxicity in Wistar Rats.

The lack of sufficient scientific evidence prompted the analytical investigation of nephroprotective potential of the silk extract of Zea mays L., which is traditionally and ethnomedicinally used for various disorders including kidney dysfunction. The present study was conducted to investigate the phytochemical analysis and demonstrate the nephroprotective potential of the methanolic silk extract of Z. mays L. using a rodent model. High-performance thin-layer chromatography (HPTLC) analysis was carried out to standardize the methanolic silk extract of Z. mays (ZME) using naringenin as a marker. The metabolite profiling of the ZME was carried out using ultrahigh-performance liquid chromatography mass spectrometry (UPLC-MS) on a monolithic capillary silica-based C18 column to identify bioactive compounds and for confirmation of the identified markers. Furthermore, for acute toxicity study, a single dose (2000 mg/kg bw) of the ZME was administered orally to Wistar rats. Also, nephrotoxicity was induced in Wistar rats by injecting diclofenac (DC) (50 mg/kg, bw, i.p.) at a single dose. The efficacy of the ZME as a nephroprotective agent was then evaluated at doses of 100, 200, and 400 mg/kg/day, bw, p.o. Furthermore, the kidney, liver, antioxidant, inflammatory, and apoptotic biochemical markers and histopathological and immunohistochemical alterations (caspase-3 and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-4 (NOX-4)) were evaluated. Phytochemical analysis by HPTLC and UPLC-MS revealed the presence of naringenin, vanillic acid, ferulic acid, gallic acid (GA), ellagic acid, quercetin, and morin, along with other bioactive constituents exhibiting multiple pharmacological properties. The acute toxicity study of the ZME showed no mortality or any clinical signs of toxicity through all the 14 days of the toxicity study at a dose of 2000 mg/kg. Also, administration of DC caused a significant elevation (P < 0.001) in kidney biochemical parameters and also caused oxidative, inflammatory, and apoptotic stress. Furthermore, DC also caused histopathological and immunohistochemical changes. Pretreatment with the ZME attenuated the elevated biochemical markers significantly at medium and high doses along with improvement in histopathological and immunohistochemical damages and showing comparable results to those of α-ketoanalogue. The present study verifies the traditional claims of Z. mays silk alleviating various kidney and related disorders by concluding the nephroprotective potential of the ZME. The nephroprotective activity of the ZME is attributed to the phytoconstituents present, acting as potent restoring antioxidants and preventing inflammatory and apoptotic cellular damages in rats. Thus, it holds promising potential in the management of nephrotoxicity.

Mycotoxin levels and characterization of natural anti-fungal phytochemicals in pearl millet (Pennisetum glaucum) from Nigeria's six agroecological zones.

This study reports levels of multiple mycotoxins across Nigeria's six agro-ecological zones and corresponding levels of natural anti-fungal phytochemicals present in pearl millet (PM). 220 representative composite samples of PM were collected for mycotoxin analysis using ultrahigh performance liquid chromatography-mass spectrometry (UHPLC-MS), and 24 were randomly selected for determination of metabolites using gas chromatography-high resolution time of flight-mass spectrometry (GC-HRTOF-MS). In total, 15 mycotoxins were detected, all with levels below the European Union (EU) permissible limits and level of aflatoxins only up to 1.34µg/kg. This is in sharp contrast to high levels of mycotoxins reported in maize samples from the same agroecological zones. Phytochemical analysis of the same samples identified a total of 88 metabolites, 30 of which are known anti-fungal properties from other previously published studies. The most common of these include methyl ester, bis (2-ethylhexyl) phthalate, and ç-tocopherol. The number of anti-fungal metabolites recovered from each sample ranged from 3 to 17 and varied widely in both number and composition across the agroecological zones. The anti-fungal metabolites may probably make PM less susceptible to fungal proliferation compared to other grains. Hence, it is worth exploring for possible sources of biological control products from PM.

The Potential Antipyretic Mechanism of Ellagic Acid with Brain Metabolomics Using Rats with Yeast-Induced Fever.

Fever is caused by an increase in the heat production process when the body is under the action of a heat source or the dysfunction of the temperature center. Ellagic acid (EA) is a polyphenol dilactone that has anti-inflammatory, anti-tumor, and antioxidant activities. Male Sprague-Dawley rats were injected yeast to reproduce an experimental fever model (150 ± 20 g), and the rectal temperature and its change values were subsequently taken 19 h later; the excessive production of interleukin-1β (IL-1β) and prostaglandin2 (PGE2) induced by yeast was regulated to normal by EA administration. Rat brain metabolomics investigation of pyrexia and the antipyretic anti-inflammatory effect of EA was performed using Ultra-High-Performance Liquid Chromatography–Mass spectrometry (UPLC-MS). Twenty-six metabolites, as potential biomarkers, significantly altered metabolites that were found in pyretic rats, and eleven metabolites, as biomarkers of the antipyretic mechanism of EA, were significantly adjusted by EA to help relieve pyrexia, which was involved in glycerophospholipid metabolism and sphingolipid metabolism, etc. In conclusion, potential metabolic biomarkers in the brain shed light on the mechanism of EA’s antipyretic effects, mainly involving metabolic pathways, which may contribute to a further understanding of the therapeutic mechanisms of fever and therapeutic mechanism of EA.

ANTIOXIDANT CAPABILITIES OF Litsea garciae BARK EXTRACTS AND THEIR RELATION TO THE PHYTOCHEMICAL COMPOSITIONS

The plant species belonging to the Litsea genus are widely investigated due to their nutritional and medicinal purposes. In this regard, this study is another similar sincere effort in which the antioxidant property and phytochemical composition of Litsea garciae (L. garciae) bark’s hexane, chloroform, methanol, and aqueous extracts were evaluated to confirm its traditional benefits. The total flavonoid content (TFC) and total phenolic content (TPC) were determined first, followed by an assessment of in vitro antioxidant activity using the DPPH and FRAP assays. The composition of the secondary metabolites was determined using Ultra-High-Performance Liquid Chromatography-Mass Spectrometry (UHPLC-MS). As a result, methanol extract was recorded to have the highest TPC value aligned with its positive appearance in phytochemical screening. Its antioxidant capacity indicated the least IC50. The results indicated that the significant free radical scavenging activity was due to the methanolic extract's high phenolic content. The secondary metabolites found in the methanol extract varied significantly according to UHPLC-MS analysis. The major phenolic compounds were found including N-trans-feruloyl-4-O-methyldopamine, N-cis-feruloyltyramine, epicatechin-(4beta-&gt;6)-epicatechin-(2beta-&gt;7,4beta-&gt;8)-epicatechin, 7-Hydroxy-3-(4-methoxyphenyl)-4-propyl-2H-1-benzopyran-2-one and 9-O-Methylneodunol. In general, the results indicate that L. garciae bark may be a promising source of novel natural compounds with antioxidative properties.

Liquid chromatography-mass spectrometry (LC-MS) based metabolomic fingerprinting in contrasting rice varieties for iron (Fe) excess.

Rice is an important cereal which is grown worldwide covering the requirement of 50% of the world population as the staple food. Diverse abiotic factors act as a limiting factor for its yield annually and can challenge sustainable productivity of the crop. Iron is an important micronutrient for plants participating in biochemical reactions including electron transport chain. Under the impact of excess iron in the rhizosphere, plants are posed with iron toxic condition. Most of the lowland rice is cultivated in submerged condition leading to accumulation of Fe2+ in the soil which is the toxic form for uptake and in turn can affect the yield of the crop. Present work is an extension of previously performed transcriptomic analysis of Northeast India lowland rice varieties under excess iron treatment using ultra-high-performance liquid chromatography-mass spectrometry (UPLC-MS). In total 1105 peaks were detected for each root and shoot. Multivariate analysis based upon principal component analysis (PCA; PCA1 and PCA2) are accounted for 70.6% of the total variation in root and 77.2% in shoot. The metabolomics profiling has indicated significant alteration in primary and secondary metabolites. Majority of the secondary metabolites like quercetin, malvin, flavanones, delphinidin are upregulated indicating their role in anti-oxidative properties during iron toxicity. On the other hand, chelators of ferric ion like mugineic acid are down regulated to avoid excess iron uptake. This work is a comparative insight on the metabolic front in case of iron excess tolerance in contrasting lowland rice varieties.

Xiao-Luo-Wan treats propylthiouracil-induced goiter with hypothyroidism in rats through the PI3K-AKT/RAS pathways based on UPLC/MS and network pharmacology

Ethnopharmacological relevanceGoiter with hypothyroidism occurs in several thyroid diseases. Xiao-Luo-Wan (XLW), which contains Scrophularia ningpoensis Hemsl., Fritillaria thunbergii Miq. and Ostrea gigas Thunberg, has been used as an effective Chinese medicine for the treatment of goiters in China for hundreds of years. Based on clinical observations and experimental studies, XLW also exerts a certain effect on hypothyroidism. However, the therapeutic mechanism of XLW remains unclear. Aim of the studyThe present study aimed to investigate the therapeutic effect of XLW on propylthiouracil (PTU)-induced goiter with hypothyroidism in rats and to uncover the underlying molecular mechanism using ultra high-performance liquid chromatography-mass spectrometry (UPLC/MS), network pharmacology, and molecular docking simulations. Materials and methodsAfter successful modeling, the remaining rats were randomly divided into a model group, an Euthyrox group, an XLW group, and a control group. The corresponding drugs were given by gavage for four consecutive weeks. The growth status was monitored, the relative thyroid weight was calculated, and the total serum T3, T4, and TSH content were detected. Hematoxylin-eosin (H&E) staining was used to observe the pathological changes in the thyroid glands. The chemical components of the XLW were identified by UPLC/MS and the putative targets of XLW were predicted using multiple databases. We performed network pharmacology based on the intersection of goiter/hypothyroidism-related targets and XLW targets. Then, we performed KEGG pathway enrichment analysis, and key targets were further screened using protein-protein interaction (PPI) networks. Finally, molecular docking was used to predict the binding ability of XLW identified components and the key targets. ResultsXLW significantly increased the levels of T3 and T4, and reduced TSH, increased body weight, and decreased swollen thyroid glands in PTU-induced rats. XLW promoted the morphological recovery of thyroid follicles and epithelial cells. Twenty-one main chemical components of XLW were identified using UPLC/MS. 270 potential gene targets of XLW and 717 known targets of goiter/hypothyroidism disease were obtained by searching the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP), Swiss Target Prediction, and UniProt databases. A total of 83 KEGG pathways were enriched with phosphatidylinositol 3-kinase-protein kinase B (PI3K-AKT) and RAS signaling pathways. PPI analysis revealed nine key targets of kinase-protein kinase B (AKT) 1, interleukin (IL) 6, vascular endothelial growth factor A (VEGFA), tumor necrosis factor (TNF), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), epidermal growth factor receptor (EGFR), GTPase HRas (HRAS), matrix metalloproteinase (MMP) 9, and heat shock protein 90 alpha family class A member 1 (HSP90AA1). Molecular docking verified which drug components had good binding ability to key targets (all ≤5 kcal/mol). ConclusionFor PTU-induced goiter with hypothyroidism in rats, XLW improves thyroid function, reduces goiter, increases body weight, and promotes the recovery of thyroid follicles and epithelial cells. The underlying molecular mechanism suggests that XLW may regulate thyroid hormone signaling by regulating the PI3K-AKT, RAS, and other signaling pathways. This study provides a pharmacological and biological basis for using XLW to treat goiter with hypothyroidism.

Metabolomics analysis of urine from rats given long-term high-protein diet using ultra-high-performance liquid chromatography-mass spectrometry

Previous studies have indicated high-protein diet (HPD) promotes weight loss and improves metabolic parameters, but most of these studies have focused on the impact of short-term, long-term effects remain unclear. In this study, male Wistar rats were fed two diets for 88 weeks: normal control diet (NCD, 20.5% of energy as protein) or HPD (30.5% of energy as protein). At 88 weeks intervention, compared to NCD rats, HPD rats had lower fat tissue and higher skeletal muscle to body weight ratio, but there were no significantly differences in body weight and food intake. To explore the mechanism underlying metabolism and diet, we further collected rat urine samples at 16, 40, 64 and 88 weeks diet treatment and analyzed metabolomics profiles using ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS). Partial least squares-discriminant analysis (PLS-DA) scores plots from ESI− or ESI+ model revealed a perfect separation between two diets at four time points. We identified 11 dramatically different metabolites (with VIP cut-off value > 1) in HPD, including 3 up-regulated and 8 down-regulated. And these 11 metabolites were identified as effective biomarkers, which were significantly related to HPD-induced metabolism related outcomes (fat tissue and skeletal muscle to body weight ratio). Our results provided vital information regarding metabolism in long-term HPD and more importantly, a few potentially promising metabolites were firstly identified which may related to metabolic responses.

Metabolomics analysis of the Finnish Geriatric Intervention Study to Prevent Cognitive Impairment and Disability (FINGER)

AbstractBackgroundThe Finnish Geriatric Intervention Study to Prevent Cognitive Impairment and Disability (FINGER) is a randomized controlled trial that showed significant benefits on cognition for a 2‐year multidomain lifestyle intervention vs regular health advice in 1260 at‐risk older individuals from the general population. The aim of this study was to investigate the metabolic effects of the intervention and their association with cognitive outcomes.MethodWe acquired metabolomics data of blood plasma on two time points (baseline and 2 years) in a set of 1089 participants (547 intervention and 542 control). A multi‐technology platform consisting of using ultra‐high performance liquid chromatography ‐ mass spectrometry (UHPLC‐MS) and 1H nuclear magnetic resonance spectroscopy (NMR) was used to generate extensive measurement coverage of the metabolome. The data was acquired using both reversed‐phase chromatography (RPC) tailored for the retention and separation of both complex and neutral lipids and hydrophilic interaction liquid chromatography (HILIC) for the retention and separation of small polar metabolites. The UHPLC‐MS untargeted data contains a total of 10,825 metabolomic features, 1244 of them currently annotated. The NMR data contains measurements of both small and macromolecules, including 112 lipoprotein fractions (derived using Bruker lipoprotein subclass analysis: BI‐LISA). Data analysis was conducted using linear mixed models.ResultSeveral baseline metabolite levels had a statistically significant association with cognition. Results will also be presented from ongoing analyses to investigate intervention effects on change in metabolites.ConclusionComplex metabolomics analyses are new in the context of multidomain lifestyle dementia prevention trials. While causality is yet to be investigated further, some of the metabolite classes and pathways observed (e.g. sphingolipids) have been previously implicated in cognitive decline and neurological disorders, suggesting early interpretable leads in the rich dataset at hand.

The Algal Polysaccharide Ulvan Induces Resistance in Wheat Against Zymoseptoria tritici Without Major Alteration of Leaf Metabolome

This study aimed to examine the ability of ulvan, a water-soluble polysaccharide from the green seaweed Ulva fasciata, to provide protection and induce resistance in wheat against the hemibiotrophic fungus Zymoseptoria tritici. Matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) analysis indicated that ulvan is mainly composed of unsaturated monosaccharides (rhamnose, rhamnose-3-sulfate, and xylose) and numerous uronic acid residues. In the greenhouse, foliar application of ulvan at 10 mg.ml–1 2 days before fungal inoculation reduced disease severity and pycnidium density by 45 and 50%, respectively. Ulvan did not exhibit any direct antifungal activity toward Z. tritici, neither in vitro nor in planta. However, ulvan treatment significantly reduced substomatal colonization and pycnidium formation within the mesophyll of treated leaves. Molecular assays revealed that ulvan spraying elicits, but does not prime, the expression of genes involved in several wheat defense pathways, including pathogenesis-related proteins (β-1,3-endoglucanase and chitinase), reactive oxygen species metabolism (oxalate oxidase), and the octadecanoid pathway (lipoxygenase and allene oxide synthase), while no upregulation was recorded for gene markers of the phenylpropanoid pathway (phenylalanine ammonia-lyase and chalcone synthase). Interestingly, the quantification of 83 metabolites from major chemical families using ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) in both non-infectious and infectious conditions showed no substantial changes in wheat metabolome upon ulvan treatment, suggesting a low metabolic cost associated with ulvan-induced resistance. Our findings provide evidence that ulvan confers protection and triggers defense mechanisms in wheat against Z. tritici without major modification of the plant physiology.