Liquid Chromatography-high Resolution Mass Spectrometry Research Articles

The potential of Sonneratia caseolaris mangrove leaves extract as a bioactive food ingredient using various water extract

Background Sonneratia caseolaris, known as the red pidada, has been widely utilized by the Indonesian. S. caseolaris leaves contain various active compounds, contributing to their popularity in the treatment of various diseases. Mangrove leaves are also known to exhibit very high antioxidant activity. This study aims to assess the antioxidant activity of S . caseolaris leaves extracted using different solvents. The resulting extract was evaluated for antioxidant activity by the 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity (DPPH) techniques. Methods Analysis of total flavonoids, total phenols, identification of active compounds with Liquid Chromatography High Resolution Mass Spectrometry (LC-HRMS), and bioinformatics were also carried out to obtain temporary conclusions about the antioxidant activity of S. caseolaris leaf extract. Results The results indicated that S. caseolaris leaves extracted with methanol and distilled water exhibited the highest antioxidant activity compared to other extracts. The analysis of total flavonoids and total phenols yielded results consistent with the antioxidant activity tests. LC-HRMS results identified three compounds in all S. caseolaris leaf extracts with antioxidant activity, namely Tempo, Choline, and Betaine. Tempo demonstrated a higher antioxidant activity than Choline and Betaine, as indicated by the binding affinity values in the bioinformatics analysis. Conclusions It is evident that S. caseolaris leaf extract has the potential to serve as an effective an antioxidant agent. Further research is needed to elucidate confirm the effectiveness of S. caseolaris leaves as an antioxidant substance, in order to utilize them as active components in food and enhance antioxidant consumption among consumers.

S-Adenosyl-l-methionine restores brain mitochondrial membrane fluidity and GSH content improving Niemann-Pick type C disease

Niemann-Pick type C (NPC) disease is a lysosomal storage disorder characterized by impaired motor coordination due to neurological defects and cerebellar dysfunction caused by the accumulation of cholesterol in endolysosomes. Besides the increase in lysosomal cholesterol, mitochondria are also enriched in cholesterol, which leads to decreased membrane fluidity, impaired mitochondrial function and loss of GSH, and has been shown to contribute to the progression of NPC disease. S-Adenosyl-l-methionine (SAM) regulates membrane physical properties through the generation of phosphatidylcholine (PC) from phosphatidylethanolamine (PE) methylation and functions as a GSH precursor by providing cysteine in the transsulfuration pathway. However, the role of SAM in NPC disease has not been investigated. Here we report that Npc1−/− mice exhibit decreased brain SAM levels but unchanged S-adenosyl-l-homocysteine content and lower expression of Mat2a. Brain mitochondria from Npc1−/− mice display decreased mitochondrial GSH levels and liquid chromatography-high resolution mass spectrometry analysis reveal a lower PC/PE ratio in mitochondria, contributing to increased mitochondrial membrane order. In vivo treatment of Npc1−/− mice with SAM restores SAM levels in mitochondria, resulting in increased PC/PE ratio, mitochondrial membrane fluidity and subsequent replenishment of mitochondrial GSH levels. In vivo SAM treatment improves the decline of locomotor activity, increases Purkinje cell survival in the cerebellum and extends the average and maximal life spam of Npc1−/− mice. These findings identify SAM as a potential therapeutic approach for the treatment of NPC disease.

Determination of per- and polyfluoroalkyl compounds in paper recycling grades using ultra-high-performance liquid chromatography–high-resolution mass spectrometry

Globally, per- and polyfluoroalkyl substances (PFAS)–related research on paper products has focused on food packaging with less consideration on the presence of PFAS at different stages of the paper recycling chain. This study analysed the prevalence of PFAS in paper grades used for the manufacture of recycled paperboard. The presence of PFAS was attributed to the use of PFAS-containing additives, consumer usage, exposure to packed goods as well as contamination during mingling, sorting, collection, and recovery of paper recycling material. Q Orbitrap mass spectrometry was used to analyse the paper samples after accelerated solvent extraction and solid phase extraction. The distribution and possible propagation of 22 PFAS were determined in pre-consumer, retail and post-consumer paper products. Post-consumer samples had the highest combined average concentration (ΣPFAS) at 213 ng/g, while the ΣPFAS in retail (159 ng/g) and pre-consumer samples (121 ng/g) was detected at lower concentrations. This study showed that waste collection and recycling protocols may influence PFAS propagation and that measures must be developed to minimise and possibly eliminate exposure opportunities.

Catabolites identification and quantification of antisense oligonucleotides by liquid chromatography - high resolution mass spectrometry (LC-HRMS)

Catabolites identification and quantification of antisense oligonucleotides by liquid chromatography - high resolution mass spectrometry (LC-HRMS)

Distribution of 2,2',5,5'-Tetrachlorobiphenyl (PCB52) Metabolites in Adolescent Rats after Acute Nose-Only Inhalation Exposure.

Inhalation of PCB-contaminated air is increasingly recognized as a route for PCB exposure. Because limited information about the disposition of PCBs following inhalation exposure is available, this study investigated the disposition of 2,2',5,5'-tetrachlorobiphenyl (PCB52) and its metabolites in rats following acute, nose-only inhalation of PCB52. Male and female Sprague-Dawley rats (50-58 days of age, 210 ± 27 g; n = 6) were exposed for 4 h by inhalation to approximately 14 or 23 μg/kg body weight of PCB52 using a nose-only exposure system. Sham animals (n = 6) were exposed to filtered lab air. Based on gas chromatography-tandem mass spectrometry (GC-MS/MS), PCB52 was present in adipose, brain, intestinal content, lung, liver, and serum. 2,2',5,5'-Tetrachlorobiphenyl-4-ol (4-OH-PCB52) and one unknown monohydroxylated metabolite were detected in these compartments except for the brain. Liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis identified several metabolites, including sulfated, methoxylated, and dechlorinated PCB52 metabolites. These metabolites were primarily found in the liver (7 metabolites), lung (9 metabolites), and serum (9 metabolites) due to the short exposure time. These results demonstrate for the first time that complex mixtures of sulfated, methoxylated, and dechlorinated PCB52 metabolites are formed in adolescent rats following PCB52 inhalation, laying the groundwork for future animal studies of the adverse effects of inhaled PCB52.

Miniaturized flexible micro-tube plasma ionization source for the effective ionization of non-easily ionizable pesticides in food with liquid chromatography/mass spectrometry

In this article, we have studied the potential of flexible microtube plasma (FμTP) as ionization source for the liquid chromatography high-resolution mass spectrometry detection of non-easily ionizable pesticides (viz. nonpolar and non-ionizable by acid/basic moieties). Phthalimide-related compounds such as dicofol, dinocap, o-phenylphenol, captan, captafol, folpet and their metabolites were studied. Dielectric barrier discharge ionization (DBDI) was examined using two electrode configurations, including the miniaturized one based on a single high-voltage (HV) electrode and a virtual ground electrode configuration (FμTP), and also the two-ring electrode DBDI configuration. Different ionization pathways were observed to ionize these challenging, non-easily ionizable nonpolar compounds, involving nucleophilic substitutions and proton abstraction, with subtle differences in the spectra obtained compared with APCI. An average sensitivity increase of 5-fold was attained compared with the standard APCI source. In addition, more tolerance with matrix effects was observed in both DBDI sources. The importance of the data reported is not just limited to the sensitivity enhancement compared to APCI, but, more notably, to the ability to effectively ionize nonpolar, late-eluting (in reverse-phase chromatography) non-ionizable compounds. Besides o-phenylphenol ([M − H]-), all the parent species were efficiently ionized through different mechanisms involving bond cleavages through the effect of plasma reagent species or its combination with thermal degradation and subsequent ionization. This tool can be used to figure out overlooked nonpolar compounds in different environmental samples of societal interest through non-target screening (NTS) strategies.

Anthropogenic-induced ecological risks on marine ecosystems indicated by characterizing emerging pollutants in Pearl River Estuary, China

Anthropogenic Contaminants of Emerging Concern (CECs) in marine environments have raised significant concerns. Yet, analyses detailing their origins, fate, and environmental effects are limited. This study employs an integrated non-target screening methodology to elucidate CECs existence across 46 sampling sites in the Pearl River Estuary (PRE) of the South China Sea. Assisted by advanced liquid chromatography-high resolution mass spectrometry, we discovered 208 chemicals in six usage categories, with pesticides (33 %) and pharmaceuticals (29 %) predominating. Several CECs drew attention for their consistent detections, profound abundance, and significant ecotoxicities. The wide detection of them at offshore sites further implies that anthropogenic activities may contribute to large-scale contamination. Meanwhile, distinct distribution patterns of CECs across PRE are evident in semi-quantitative results, indicating regional anthropogenic influences. Identified transformation products may establish a novel and non-negligible negative contribution to ecology through elevated environmental toxicities, exemplified by HMMM and atrazine. Based on the ecological risks, we compiled a prioritized list of 21 CECs warranting intensified scrutiny. Our findings indicate the introduction of various CECs into the South China Sea via PRE, emphasizing the urgent necessity for ongoing surveillance of discharged CECs at estuary areas and assessment of their marine ecological consequences.

Investigation of pectin deficiency in modulating the bioflavonoid profile of orange processing waste: A sustainable valorization of industrial waste

Orange processing waste (OPW) generated by the processing of oranges, as well as other citrus fruits, is a major source of pectin in the market nowadays. The residues generated during the pectin extraction process may contain many phytochemicals, including flavonoids. We use state-of-the-art techniques such as liquid chromatography high-resolution mass spectrometry (LC-HRMS/MS) and feature-based molecular network (FBMN) to annotate the flavonoids in OPWs. In particular, four flavonoids, hesperidin, naringin, diosmin, and hesperetin were quantified in the samples by LC-TDQ-MS. In total, 32 flavonoids from different classes were annotated, of which 16 were polymethoxylated flavonoids, 13 were flavonoid glycosides and 3 were flavanone aglycones. The results showed that flavonoid glycosides remain in high concentrations in OPWs from pectin factories even after pectin extraction by harsh conditions. The results show an exciting opportunity to harness the untapped potential of pectin factory waste as a renewable source for the extraction of glycoside flavonoids.

Interplatform comparison between three ion mobility techniques for human plasma lipid collision cross sections

The implementation of ion mobility spectrometry (IMS) in liquid chromatography-high-resolution mass spectrometry (LC-HRMS) workflows has become a valuable tool for improving compound annotation in metabolomics analyses by increasing peak capacity and by adding a new molecular descriptor, the collision cross section (CCS). Although some studies reported high repeatability and reproducibility of CCS determination and only few studies reported good interplatform agreement for small molecules, standardized protocols are still missing due to the lack of reference CCS values and reference materials.We present a comparison of CCS values of approximatively one hundred lipid species either commercially available or extracted from human plasma. We used three different commercial ion mobility technologies from different laboratories, drift tube IMS (DTIMS), travelling wave IMS (TWIMS) and trapped IMS (TIMS), to evaluate both instrument repeatability and interlaboratory reproducibility. We showed that CCS discrepancies of 0.3% (average) could occur depending on the data processing software tools. Moreover, eleven CCS calibrants were evaluated yielding mean RSD below 2% for eight calibrants, ESI Low concentration tuning mix (Tune Mix) showing the lowest RSD (< 0.5%) in both ion modes. Tune Mix calibrated CCS from the three different IMS instruments proved to be well correlated and highly reproducible (R2 > 0.995 and mean RSD ≤ 1%). More than 90% of the lipid CCS had deviations of less than 1%, demonstrating high comparability between techniques, and the possibility to use the CCS as molecular descriptor. We highlighted the need of standardized procedures for calibration, data acquisition, and data processing. This work demonstrates that using harmonized analytical conditions are required for interplatform reproducibility for CCS determination of human plasma lipids.

Methanolic Extract of Neurocalynx calcinus for Promoting Wound Healing: Characterization by Ultra-High-Performance Liquid Chromatography High-Resolution Mass Spectrometry

Methanolic Extract of Neurocalynx calcinus for Promoting Wound Healing: Characterization by Ultra-High-Performance Liquid Chromatography High-Resolution Mass Spectrometry

Predicting the Activity of Unidentified Chemicals in Complementary Bioassays from the HRMS Data to Pinpoint Potential Endocrine Disruptors.

The majority of chemicals detected via nontarget liquid chromatography high-resolution mass spectrometry (HRMS) in environmental samples remain unidentified, challenging the capability of existing machine learning models to pinpoint potential endocrine disruptors (EDs). Here, we predict the activity of unidentified chemicals across 12 bioassays related to EDs within the Tox21 10K dataset. Single- and multi-output models, utilizing various machine learning algorithms and molecular fingerprint features as an input, were trained for this purpose. To evaluate the models under near real-world conditions, Monte Carlo sampling was implemented for the first time. This technique enables the use of probabilistic fingerprint features derived from the experimental HRMS data with SIRIUS+CSI:FingerID as an input for models trained on true binary fingerprint features. Depending on the bioassay, the lowest false-positive rate at 90% recall ranged from 0.251 (sr.mmp, mitochondrial membrane potential) to 0.824 (nr.ar, androgen receptor), which is consistent with the trends observed in the models' performances submitted for the Tox21 Data Challenge. These findings underscore the informativeness of fingerprint features that can be compiled from HRMS in predicting the endocrine-disrupting activity. Moreover, an in-depth SHapley Additive exPlanations analysis unveiled the models' ability to pinpoint structural patterns linked to the modes of action of active chemicals. Despite the superior performance of the single-output models compared to that of the multi-output models, the latter's potential cannot be disregarded for similar tasks in the field of in silico toxicology. This study presents a significant advancement in identifying potentially toxic chemicals within complex mixtures without unambiguous identification and effectively reducing the workload for postprocessing by up to 75% in nontarget HRMS.

Exposure to per- and polyfluoroalkyl substances and high-throughput proteomics in Hispanic youth

BackgroundStrong epidemiological evidence shows positive associations between exposure to per- and polyfluoroalkyl substances (PFAS) and adverse cardiometabolic outcomes (e.g., diabetes, hypertension, and dyslipidemia). However, the underlying cardiometabolic-relevant biological activities of PFAS in humans remain largely unclear. AimWe evaluated the associations of PFAS exposure with high-throughput proteomics in Hispanic youth. Material and MethodsWe included 312 overweight/obese adolescents from the Study of Latino Adolescents at Risk (SOLAR) between 2001 and 2012, along with 137 young adults from the Metabolic and Asthma Incidence Research (Meta-AIR) between 2014 and 2018. Plasma PFAS (i.e., PFOS, PFOA, PFHxS, PFHpS, PFNA) were quantified using liquid-chromatography high-resolution mass spectrometry. Plasma proteins (n = 334) were measured utilizing the proximity extension assay using an Olink Explore Cardiometabolic Panel I. We conducted linear regression with covariate adjustment to identify PFAS-associated proteins. Ingenuity Pathway Analysis, protein-protein interaction network analysis, and protein annotation were used to investigate alterations in biological functions and protein clusters. ResultsResults after adjusting for multiple comparisons showed 13 significant PFAS-associated proteins in SOLAR and six in Meta-AIR, sharing similar functions in inflammation, immunity, and oxidative stress. In SOLAR, PFNA demonstrated significant positive associations with the largest number of proteins, including ACP5, CLEC1A, HMOX1, LRP11, MCAM, SPARCL1, and SSC5D. After considering the mixture effect of PFAS, only SSC5D remained significant. In Meta-AIR, PFAS mixtures showed positive associations with GDF15 and IL6. Exploratory analysis showed similar findings. Specifically, pathway analysis in SOLAR showed PFOA- and PFNA-associated activation of immune-related pathways, and PFNA-associated activation of inflammatory response. In Meta-AIR, PFHxS-associated activation of dendric cell maturation was found. Moreover, PFAS was associated with common protein clusters of immunoregulatory interactions and JAK-STAT signaling in both cohorts. ConclusionPFAS was associated with broad alterations of the proteomic profiles linked to pro-inflammation and immunoregulation. The biological functions of these proteins provide insight into potential molecular mechanisms of PFAS toxicity.

Urine Metabolite Profiles after the Consumption of a Low- and a High-Digestible Protein Meal, and Comparison of Urine Normalization Techniques.

In the context of dietary transition toward plant proteins, it is necessary to ensure protein security in populations. It would thus be of interest to identify biomarkers of altered protein digestibility in populations. We examined the association between urinary metabolites and the acute intake of low- or highly digestible protein in healthy volunteers. The urine samples were collected before and 9 h after the ingestion of a meal containing either no protein, zein (low-digestible) or whey protein isolate (highly digestible). The liquid chromatography-high resolution mass spectrometry metabolomics approach was used for the profiling of the urinary metabolites. For the standardization of metabolomics data sets, osmolality-based, standard normal variates (SNV) and probabilistic quotient normalization (PQN) techniques were used. The ANOVA-based factorial method, AComDim_ICA, was used for chemometrics analysis. The osmolality adjustment has a beneficial effect and the subsequent mathematical normalization improves the chemometric analysis further. Some changes in the urinary metabolomes were observed 9 h after the meal in the three groups. However, there was no difference in the urine metabolome between groups. No biomarker of protein digestibility can be identified after the ingestion of a single meal, even when marked differences in the digestion efficiency of protein have been observed.

Abstract 3745: Metabolomic profiling of lipid and metabolite in cirrhosis patients with and without hepatocellular carcinoma

Abstract Early detection of hepatocellular carcinoma (HCC) is crucial for improving patient outcomes, as curative treatments are most effective at the early stages of the disease. The lack of robust biomarkers, particularly a non-invasive diagnostic tool, precludes significant improvement of clinical outcomes for HCC patients. Serum metabolites are one of the best non-invasive means for determining patient prognosis. Established biomarkers for HCC including alpha-fetoprotein (AFP), have shown suboptimal performance in early disease stages. This study aimed to develop a combined metabolite and lipid panel to differentiate early-stage HCC from cirrhosis. Unbiased metabolomic and lipidomic analyses of serum samples were performed for 28 and 30 patients with early HCC and cirrhosis, respectively. We developed an unbiased high-resolution liquid chromatography mass spectrometry (LC-MS) based metabolic profiling platform and evaluated differences in the serum global metabolome and lipidome. This method enabled the detection of over 1200 metabolites and up to 800 lipid molecules in this select cohort. We identified 124 metabolites and 246 lipids that were upregulated and 208 metabolites and 73 lipids that were downregulated in HCC compared to cirrhosis. Using multiomic integrative analysis, we identified the overlap between differentially expressed metabolites and lipid and previously published transcriptomic signatures and illustrate that the overlapping signature associates with clinical disease progression. Lastly, we leveraged machine learning models to identify a minimal panel of lipids and metabolites that accurately distinguish between cirrhosis patients with HCC and without HCC. The best performing classifier was derived using Support Vector Machines, achieving a median AUC of 0.98 over 100 cross-validation iterations, and we showed that we need as little as 12 metabolite and lipids for effective discrimination between cirrhosis and early HCC. Citation Format: Hannah Powell, Elisa Ruiz, Sandra L. Grimm, Omar Najjar, Luis Olivares, Michael Scheurer, Hashem B. El Serag, Cristain Coarfa, Salma Kaochar. Metabolomic profiling of lipid and metabolite in cirrhosis patients with and without hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3745.

Unraveling varietal differences and nutraceutical potentials of Manjari Medika grape hybrid and its parents: An untargeted metabolomics study

The study presents a robust, reliable, and precise classification model that accurately compares the metabolite composition of an Indian grape hybrid named Manjari Medika (MM) to its parental lines, Pusa Navrang (PN) and Flame Seedless (FS). The objective was to evaluate the impact of PN and FS on the MM metabolome, and investigate the nutraceutical potential of these three grape varieties. By employing a liquid chromatography-high resolution mass spectrometry based untargeted approach, the methanolic extracts of these grapes were screened. The metabolites were identified by matching with globally available databases and were further subjected to multivariate statistical analysis. The study identified 427 metabolites, out of which 410 compounds were common across all three varieties. The MM cultivar revealed 73 distinct compounds. The chemoinformatics analysis identified 21 metabolites as distinguishing biomarkers. In MM, the elevated relative abundance of the potentially bioactive metabolites suggests its suitability as a potential functional food. From the statistical analysis, it is hypothesized that the anthocyanin profile and drought tolerance may be influenced by PN, while FS could potentially contribute to enhanced shelf life. This study could help grape breeders map inheritance pattern of bioactive compounds and identify the best parents and progenies to create robust varieties with rich metabolite profiles.

In silico and Biochemical Approach for Isolated, Purified, and Characterized Protease Inhibitor from Limonia acidissima

Proteases produced by various bacterial and fungal pathogens are associated with pathogenicity and cause septic hazards. Thereby inhibition of such proteases by protease inhibitors from natural sources is effective strategy to combat infectious diseases. Protease inhibitors originating from plant source are abundant in nature and play an important role in defense mechanism against virulent microbes. Many protease inhibitors can be isolated and purified from plant sources and formulated as therapeutic drugs to combat human diseases. In spite of their applications in biomedicines, it is necessary to explore the novel biochemical characteristics of protease inhibitors with improved efficacy. The objective of the current study was extraction, purification and biochemical characterization of protease inhibitor from L. acidissima. The protease inhibitor was isolated from L. acidissima by buffer extraction method and confirmed for anti-protease activity using standard trypsin assay. The inhibitor was found to be active between pH ranging from 5-9 with maximal activity at pH 7. The inhibitor showed thermal stability at a temperature of 50-60°C and retained 70% activity at 50°C for 2 hr. Inhibitor activity was enhanced by thermal stabilizers glycine, urea, Calcium chloride (CaCl2), glycerol, and sucrose at 50°C; metal ions Cu2+ and Al3+ and detergents like Sodium dodecyl sulfate (SDS), Triton-X 100, Tween-80. Protease inhibitor was also stable in the presence of oxidizing and reducing agents Dimethyl sulfoxide (DMSO), Hydrogen peroxide (H2O2), β-mercaptoethanol, and Sodium Thioglycolate. Thus, optimization and characterization studies indicate that the isolated inhibitor is active under natural conditions. Protease inhibitor showed positive results for anticancer, anti-diabetic and anti-inflammatory activities indicating the therapeutic potential of inhibitor molecule. Further identification and separation of bioactive molecules from the buffer extract of L. acidissima was carried out by Flash chromatography followed by Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis that revealed the presence of Diisobutylphthalate as bioactive phytoconstituent. Docking studies performed revealed the anti-diabetic, anti-inflammatory, and anti-cancer potential of Diisobutylphthalate. Thus, in silico studies support the activity of our isolated protease inhibitor to ensure a promising future in the development of therapeutics.

LC-HRMS and NMR studies for the characterization of degradation impurities of ubrogepant along with the in silico approaches for the prediction of degradation and toxicity

Ubrogepant is the first oral calcitonin gene-related peptide (CGRP) receptor antagonist which is used for the acute treatment of migraine in adults. The present study employs liquid chromatography-high resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance spectroscopy (NMR) techniques for the identification and characterization of degradation impurities of ubrogepant. The forced degradation study of ubrogepant was performed as per the International Council for Harmonisation (ICH) Q1A and Q1B guidelines. The in silico degradation profile of ubrogepant was predicted by Zeneth. It was observed that ubrogepant was labile to acidic hydrolysis, basic hydrolysis, and oxidative degradation conditions (H2O2), although it was stable in neutral hydrolysis and photolytic (UV light and visible light) conditions. Eight degradation impurities were formed, which were separated on reversed-phase HPLC with a gradient program on an InertSustain C8 column (4.6 × 250 mm, 5 µm) using 10 mM ammonium formate (pH unadjusted) and acetonitrile as the mobile phase. The structures of all the degradation impurities were characterized using the exact masses obtained from the HRMS/MS. Further, NMR studies were conducted on two major degradation impurities (UB-4 and UB-7). A plausible mechanism was proposed to support the structures of all the degradation impurities of UBR. In silico toxicity and mutagenicity assessment were done by DEREK Nexus, SARAH Nexus, and ProTox-II.

Resultant compound from sublimation test for Gentianae Radix in Japanese Pharmacopoeia was 5-(hydroxymethyl)furfural.

Gentianae Radix, an herbal medicine, has been used as a gastrointestinal drug in Japan. In the Japanese Pharmacopoeia 18th Revision, the sublimation test is specified as an identification test for Gentianae Radix. The compound obtained in this sublimation test was believed to be gentisin, a xanthone family compound. However, the compound we identified using liquid chromatography-high-resolution mass spectrometry (LC-HRMS) and 1H- and 13C-NMR was 5-(hydroxymethyl)furfural (5-HMF). The same compound was found to be a sublimate of Gentianae Scabrae Radix and Gentianae Macrophyllae Radix, belonging to the same genus as Gentianae Radix. These results indicate the necessity to revise the identification test for Gentianae Radix to a more unique method.

Unveiling the Diversity and Modifications of Short Peptides in Buthus martensii Scorpion Venom through Liquid Chromatography-High Resolution Mass Spectrometry.

More recently, short peptides in scorpion venom have received much attention because of their potential for drug discovery. Although various biological effects of these short peptides have been found, their studies have been hindered by the lack of structural information especially in modifications. In this study, small peptides from scorpion venom were investigated using high-performance liquid chromatography high-resolution mass spectrometry followed by de novo sequencing. A total of 156 sequences consisting of 2~12 amino acids were temporarily identified from Buthus martensii scorpion venom. The identified peptides exhibited various post-translational modifications including N-terminal and C-terminal modifications, in which the N-benzoyl modification was first found in scorpion venom. Moreover, a short peptide Bz-ARF-NH2 demonstrated both N-terminal and C-terminal modifications simultaneously, which is extremely rare in natural peptides. In conclusion, this study provides a comprehensive insight into the diversity, modifications, and potential bioactivities of short peptides in scorpion venom.

Abrus precatorius Leaf Extract Stimulates Insulin-mediated Muscle Glucose Uptake: In vitro Studies and Phytochemical Analysis.

Diabetes mellitus, linked with insulin resistance and hyperglycaemia, is a leading cause of mortality. Glucose uptake through glucose transporter type 4, especially in skeletal muscle, is crucial for maintaining euglycaemia and is a key pathway targeted by antidiabetic medication. Abrus precatorius is a medicinal plant with demonstrated antihyperglycaemic activity in animal models, but its mechanisms are unclear.This study evaluated the effect of a 50% ethanolic (v/v) A.precatorius leaf extract on (1) insulin-stimulated glucose uptake and (2) related gene expression in differentiated C2C12 myotubes using rosiglitazone as a positive control, and (3) generated a comprehensive phytochemical profile of A.precatorius leaf extract using liquid chromatography-high resolution mass spectrometry to elucidate its antidiabetic compounds. A.precatorius leaf extract significantly increased insulin-stimulated glucose uptake, and insulin receptor substrate 1 and Akt substrate of 160 kDa gene expression; however, it had no effect on glucose transporter type 4 gene expression. At 250 µg/mL A.precatorius leaf extract, the increase in glucose uptake was significantly higher than 1 µM rosiglitazone. Fifty-five phytochemicals (primarily polyphenols, triterpenoids, saponins, and alkaloids) were putatively identified, including 24 that have not previously been reported from A.precatorius leaves. Abrusin, precatorin I, glycyrrhizin, hemiphloin, isohemiphloin, hispidulin 4'-O-β-D-glucopyranoside, homoplantaginin, and cirsimaritin were putatively identified as known major compounds previously reported from A.precatorius leaf extract. A.precatorius leaves contain antidiabetic phytochemicals and enhance insulin-stimulated glucose uptake in myotubes via the protein kinase B/phosphoinositide 3-kinase pathway by regulating insulin receptor substrate 1 and Akt substrate of 160 kDa gene expression. Therefore, A.precatorius leaves may improve skeletal muscle insulin sensitivity and hyperglycaemia. Additionally, it is a valuable source of bioactive phytochemicals with potential therapeutic use for diabetes.