Determination of α-methylenecyclopropylglycine in Shahi and China litchi cultivars at three different maturity stages: A quantitative study using liquid chromatography tandem mass spectrometry.

This study presents the contents of α-methylenecyclopropylglycine, a potentially toxic amino acid, in the peel, pulp and seed fractions of two well-known litchi varieties, namely Shahi and China, over a span of three harvest-seasons. For analysing α-methylenecyclopropylglycine, an LC-MS/MS-based method was validated. The method-accuracies fell within 75-110 % (RSD, <15 %) at 0.1 mg/kg (LOQ) and higher levels. A comparative evaluation of the results in peel, pulp and seed at 30 days before harvest (DBH), 15-DBH, and edible-ripe stage revealed that α-methylenecyclopropylglycine content increased as the litchi seeds grew towards maturity, regardless of the cultivar. In arils, at maturity, the concentration of α-methylenecyclopropylglycine ranged from not-detected to 11.7 µg/g dry weight. The Shahi cultivar showed slightly higher α-methylenecyclopropylglycine content in comparison to China litchi. This paper presents the first known analysis of combined seasonal data on different fruit components at various growth stages for the two chosen litchi cultivars grown in India.

Evidence to support cultivated fruiting body of Ophiocordyceps sinensis (Ascomycota)'s role in relaxing airway smooth muscle.

ETHNOPHARMACOLOGICAL RELEVANCE: Ophiocordyceps sinensis (O. sinensis) is a genus of Ascomycete fungus that is endemic to the alpine meadows of the Tibetan Plateau and adjoining Himalayas. It has been used traditionally as a tonic to improve respiratory health in ancient China as well as to promote vitality and longevity. Bioactive components found in O. sinensis such as adenosine, cordycepin, 3-deoxyadenosine, L-arginine and polysaccharides have gained increasing interest in recent years due to their antioxidative and other properties, which include anti-asthmatic, antiviral, immunomodulation and improvement of general health. AIM OF THE STUDY: This study's primary aim was to investigate the effect of a cultivated fruiting body of O. sinensis strain (OCS02®) on airways patency and the secondary focus was to investigate its effect on the lifespan of Caenorhabditis elegans. MATERIALS AND METHODS: A cultivated strain, OCS02®, was employed and the metabolic profile of its cold-water extract (CWE) was analysed through liquid chromatography-mass spectrometry (LC-MS). Organ bath approach was used to investigate the pharmacological properties of OCS02® CWE when applied on airway tissues obtained from adult male Sprague-Dawley rats. The airway relaxation mechanisms of OCS02® CWE were explored using pharmacological tools, where the key regulators in airway relaxation and constriction were investigated. For the longevity study, age-synchronised, pos-1 RNAi-treated wild-type type Caenorhabditis elegans at the L4 stage were utilised for a lifespan assay. RESULTS: Various glycopeptides and amino acids, particularly a high concentration of L-arginine, were identified from the LC-MS analysis. In airway tissues, OCS02® CWE induced a significantly greater concentration-dependent relaxation when compared to salbutamol. The relaxation response was significantly attenuated in the presence of NG-Nitro-L-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ) and several K+ channel blockers. The longevity effect induced by OCS02® CWE (5 mg/mL and above) was observed in C. elegans by at least 17%. CONCLUSIONS: These findings suggest that the airway relaxation mechanisms of OCS02® CWE involved cGMP-dependent and cGMP-independent nitric oxide signalling pathways. This study provides evidence that the cultivated strain of OCS02® exhibits airway relaxation effects which supports the traditional use of its wild O. sinensis in strengthening respiratory health.

Global Metabolomics Using LC-MS for Clinical Applications.

Metabolomics can be used for a multitude of purposes, including monitoring of treatment effects and for increasing the knowledge of the pathophysiology of a wide range of diseases. Global (commonly referred to as "untargeted") metabolomics is hypothesis-generating and provides the opportunity to discover new biomarkers. Being versatile and having a high degree of selectivity and sensitivity, liquid chromatography-mass spectrometry (LC-MS) is the most common technique applied for metabolomics. We here present our global metabolomics LC-electrospray ionization-MS/MS method. The sample preparation procedures for plasma, serum, dried blood spots, urine, and cerebrospinal fluid are simple and nonspecific to reduce the risk of analyte loss. The method is based on reversed-phase chromatography using a diphenyl column. The high-resolution Q Exactive Orbitrap MS with data-dependent acquisition provides MS/MS spectra of a wide range of analytes. Our method covers a large part of the metabolome regarding hydrophobicity and compound class.

Distinguishing Artifactual Fatty Acid Dimers from Fatty Acid Esters of Hydroxy Fatty Acids in Untargeted LC-MS Pipelines.

Untargeted metabolomics is a powerful profiling tool for the discovery of possible biomarkers of disease onset and progression. Analytical pipelines applying liquid chromatography (LC) and mass spectrometry (MS)-based methods are widely used to survey a broad range of metabolites within various metabolic pathways, including organic acids, amino acids, nucleosides, and lipids. Accurate and complete identification of putative metabolites is an ongoing challenge in untargeted metabolomics studies. Highly sensitive instrumentation can result in the detection of adduct and fragment ions that form reproducibly and contain identifiable ions that are difficult to distinguish from metabolic pathway intermediates, which may result in false-positive identification. At concentrations as low as 10 µM, free fatty acids have been found to form homo- and heterodimers in untargeted metabolomics pipelines that resemble the lipid class fatty acid esters of hydroxy fatty acids (FAHFAs), resulting in misidentification. This chapter details a protocol for LC-MS-based untargeted metabolomics using hydrophilic interaction chromatography (HILIC) that specifically aids in distinguishing artifactual fatty acid dimers from endogenous FAHFAs.

LC-MS/MS Quantification of Endocannabinoids in Tissues.

Endocannabinoids (ECBs) are lipid-derived endogenous molecules with important physiological roles such as regulation of energy balance, immunity, or neural development. Quantitation of ECBs helps better understand their physiological role and modulation of biological processes. This chapter presents the simultaneous quantification of 14 ECBs and related molecules in the brain, liver, and muscle, as well as white and brown adipose tissue using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The dynamic range of the method has been tuned to cover the endogenous concentrations of these analytes given the fact that they are endogenously present at different orders of magnitude. Specifically, three groups are established: 0.5-5000 ng/mL for 2-oleoyl- and 2-linoleoylglycerol and arachidonic acid, 0.05-500 ng/mL for 2-arachidonoylglycerol, and 0.0005-0.5 ng/mL for anandamide, palmitoyl-, palmitoleoyl-, stearoyl-, oleoyl-, linoleoyl-, alpha-linolenoyl-, dihomo-gamma-linolenoyl-, docosahexaenoyl-, and pentadecanoylethanolamide.

Metabolomics of Chinese Hamster Ovary Cells.

Increasing demand of protein biotherapeutics produced using Chinese hamster ovary (CHO) cell lines necessitates improvement in the production yield of the bioprocess. Various cell engineering, improved media formulation and process-design based approaches utilizing the power of OMICS technologies, specifically, genomics and proteomics, have been employed; however, the potential of metabolomics largely remains unexplored. Metabolomics enables the detection, identification, and/or quantitation of small molecules, commonly known as metabolites, in and around the cells and may help to unlock the cellular molecular mechanism(s) that regulates cell growth and productivity in the bioprocess and improves cellular performance during the bioprocess. Currently, liquid chromatography (LC)/gas chromatography (CG)- coupled with mass-spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy are the most commonly used approaches for metabolomics. Therefore, in this chapter, we have discussed the standard procedures of investigating CHO metabolites using LC/GC-MS and/or NMR-based approaches.

Analytical Tools for HCP Detection, Identification, and Quantitation.

Identifying and quantifying host cell proteins (HCPs) are crucial in developing and manufacturing biopharmaceutical products. ELISA has been used widely for identifying and quantifying HCPs in biopharmaceutical products. Liquid chromatography-mass spectrometry (LC-MS) has recently emerged as an orthogonal tool for HCP analysis, providing comprehensive and quantitative information on individual HCPs. This chapter explores the advancements in LC-MS/MS methodologies for HCP analysis, including sample preparation, data acquisition modes, and data analysis strategies. It also discusses the challenges of HCP identification and quantitation, and recent innovations to overcome these limitations. The application of LC-MS/MS methods in HCP profiling holds significant promise for improving process control and ensuring product safety in biopharmaceutical manufacturing.

Spatial metabolomics, LC-MS and RNA-Seq reveal the effect of red and white muscle on rabbit meat flavor.

Meat quality is a key factor influencing consumer purchasing decisions. Muscle composition consists of various types of myofibers (type I and type IIa, IIb, IIx myofibers), and the relative composition of fiber types has a significant impact on the overall biochemical properties and flavor of fresh meat. However, the relationship between biochemical changes in myofibers and their impact on meat quality remains underexplored. In this study, we compared the differences in meat quality by examining different muscles in rabbits, each containing different muscle fiber types. We focused on the adductor (ADD) and semitendinosus (ST) as our research subjects and investigated skeletal muscle metabolism at the individual myofibers level using Spatial metabolomics. Additionally, we utilized LC-MS and RNA-Seq to explore the molecular mechanisms underlying the metabolic differences between red and white muscle fibers. Our findings demonstrated that variations in myofiber composition significantly influenced meat color, pH, water content, and drip loss. Spatial metabolomics analysis identified 22 unique red and white muscle fingerprint metabolites, while LC-MS analysis revealed 123 differential metabolites, and these differential metabolites were mainly enriched in the pathways of ABC transporters, Biosynthesis of amino acids, glutathione metabolism, and arginine biosynthesis. To further elucidate the molecular mechanism of differential metabolism in ADD and ST, we identified 2248 differentially expressed genes (DEGs) by RNA-Seq and then combined DEGs with DMs for joint analysis. We found that red muscle exhibited higher levels of metabolites such as L-glutamic acid, glutathione, ascorbate, ornithine, oxidized glutathione, gamma-L-glutamyl-L-cysteine, cysteinylglycine, fumaric acid, gamma-aminobutyric acid. Additionally, related metabolic genes such as MGST1, ODC1, MGST3 and PRDX6 were highly expressed in ST muscle. These metabolites and genes were enriched in the glutathione and nicotinamide pathways, and had significant effects on meat color and drip loss. Moreover, red muscle contained more flavor compounds and nutrients, including adenosine monophosphate (AMP), ornithine, citrulline, taurine, acetyl phosphate, L-glutamic acid metabolites, as well as taurine and hypotaurine metabolites. Our results demonstrate that fresh meat with a higher proportion of red muscle fibers exhibited superior meat quality, enhanced flavor, and higher nutrient content. Furthermore, red muscle contains more antioxidant metabolites that can effectively prevent meat oxidation during the production process.

Towards prediction of maturation-dependent kokumi taste in cheese by comprehensive high throughput quantitation of glutamyl dipeptides.

The study focuses on the comprehensive analysis of glutamyl dipeptides in cheese, particularly their formation during the cheese ripening process and the influence of various factors, such as origin, the use of various mold cultures, and cheese types. For the first time, all three subgroups of glutamyl dipeptides, namely α-Glu-X, X-Glu, and γ-Glu-X, are covered in a comprehensive analytical LC-MS/MS method offering robust quantitation of all 56 glutamyl dipeptides. The workflow includes a simplified extraction protocol and an optimized separation of the analytes on the stationary phase. Validation experiments demonstrate the method's reliability, including repeatability, detection limits, and recovery. The comprehensive analysis of all glutamyl dipeptides in 122 cheese samples with ripening times between 2 weeks and 15 years shows a strong increase in all peptide classes with prolonged ripening and particularly in the presence of mold.

Biomarker Discovery via N-Glycoproteomics.

Posttranslational modifications (PTMs) of proteins regulate several biological processes, and investigating their diversity is crucial for understanding the mechanisms of cell regulation. Glycosylation is one of the most complex posttranslational modifications that control fundamental cellular processes such as protein folding, protein trafficking, host-pathogen interactions, cell adhesion, and cytokine receptor signaling networks. N-linked glycosylation denotes the attachment of glycans (oligosaccharides) to a nitrogen atom of asparagine (N) residues in the consensus motif Asn-X-Ser/Thr (NXS/T), where X is any amino acid except proline. Therefore, mutations in this posttranslational modification (i.e., N-glycosylation) site cause many human genetic diseases, including cancer. In the past decade, high-throughput quantitative proteome profiling tools have significantly renewed our interest in discovering novel cancer diagnostic or prognostic biomarkers through the simultaneous examination of the enormous amount of high-quality data of thousands of proteins and genes in complex biological systems. In this chapter, we describe how aberrant N-linked glycopeptides could be selectively identified as novel single tumor markers through the use of mass spectrometry (MS)-based proteomics, also known as Solid-phase extraction of N-glycopeptides (SPEG), and reasonable hypotheses that have the potential capacity to revolutionize biomarker discovery and bring those markers to the clinic as early as possible.

Direct analysis in real time mass spectrometry (DART-MS/MS) for rapid urine opioid detection in a clinical setting.

BACKGROUND AND AIMS: Current laboratory methods for opioid detection involve an initial screening with immunoassays which offers efficient but non-specific results and a subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmation which offers accurate results but requires extensive sample preparation and turnaround time. Direct Analysis in Real Time (DART) tandem mass spectrometry is evaluated as an alternative approach for accurate opioid detection with efficient sample preparation and turnaround time. MATERIALS AND METHODS: DART-MS/MS was optimized by testing the method with varying temperatures, operation modes, extraction methods, hydrolysis times, and vortex times. The method was evaluated for 12 opioids by testing the analytical measurement range, percent carryover, precision studies, stability, and method-to-method comparison with LC-MS/MS. RESULTS: DART-MS/MS shows high sensitivity and specificity for the detection of 6-acetylmorphine, codeine, hydromorphone, oxymorphone, hydrocodone, naloxone, buprenorphine, norfentanyl, and fentanyl in urine samples. However, its performance was suboptimal for norbuprenorphine, morphine and oxycodone. CONCLUSION: In this proof-of-concept study, DART-MS/MS is evaluated for its rapid quantitative definitive testing of opioids drugs in urine. Further research is needed to expand its application to other areas of drug testing.

Application of Proteomics Technology Based on LC-MS Combined with Western Blotting and Co-IP in Antiviral Innate Immunity.

As an interferon-stimulating factor protein, STING plays a role in the response and downstream liaison in antiviral natural immunity. Upon viral invasion, the immediate response of STING protein leads to a series of changes in downstream proteins, which ultimately leads to an antiviral immune response in the form of proinflammatory cytokines and type I interferons, thus triggering an innate immune response, an adaptive immune response in vivo, and long-term protection of the host. In the field of antiviral natural immunity, it is particularly important to rigorously and sequentially probe the dynamic changes in the antiviral natural immunity connector protein STING caused by the entire anti-inflammatory and anti-pathway mechanism and the differences in upstream and downstream proteins. Traditionally, proteomics technology has been validated by detecting proteins in a 2D platform, for which it is difficult to sensitively identify changes in the nature and abundance of target proteins. With the development of mass spectrometry (MS) technology, MS-based proteomics has made important contributions to characterizing the dynamic changes in the natural immune proteome induced by viral infections. MS analytical techniques have several advantages, such as high throughput, rapidity, sensitivity, accuracy, and automation. The most common techniques for detecting complex proteomes are liquid chromatography (LC) and mass spectrometry (MS). LC-MS (Liquid Chromatography-Mass Spectrometry), which combines the physical separation capability of LC and the mass analysis capability of MS, is a powerful technique mainly used for analyzing the proteome of cells, tissues, and body fluids. To explore the combination of traditional proteomics techniques such as Western blotting, Co-IP (co-Immunoprecipitation), and the latest LC-MS methods to probe the anti-inflammatory pathway and the differential changes in upstream and downstream proteins induced by the antiviral natural immune junction protein STING.

Identifying an Abnormal Phosphorylated Adaptor by Viral Kinase Using Mass Spectrometry.

Mass spectrometers are widely used to identify protein phosphorylation sites. The process usually involves selective isolation of phosphoproteins and subsequent fragmentation to identify both the peptide sequence and phosphorylation site. Immunoprecipitation could capture and purify the protein of interest, greatly reducing sample complexity before submitting it for mass spectrometry analysis. This chapter describes a method to identify an abnormal phosphorylated site of the adaptor protein by a viral kinase through immunoprecipitation followed by LC-MS/MS.

Catalytic detoxification of mitoxantrone by graphitic carbon nitride (g-C3N4) supported Fe/Pd bimetallic nanoparticles.

The overuse of antibiotics and antitumor drugs has resulted in more and more extensive pollution of water bodies with organic drugs, causing detrimental ecological effects, which have attracted attention towards effective and sustainable methods for antibiotics and antitumor drug degradation. Here, the hybrid nanomaterial (g-C3N4@Fe/Pd) was synthesized and used to remove a kind of both an antibiotic and antitumor drug named mitoxantrone (MTX) with 92.0% removal efficiency, and the MTX removal capacity is 450 mg/g. After exposing to the hybrid material the MTX aqueous solution changed color from dark blue to lighter progressively, and LC-UV results of residual solutions show that a new peak at 3.0 min (MTX: 13.2 min) after removal by g-C3N4@Fe/Pd appears, with the simultaneous detection of intermediate products indicating that g-C3N4@Fe/Pd indeed degrades MTX. Detailed mass spectrometric analysis suggests that the nuclear mass ratio decreased from 445.2 (M+1H) to 126.0 (M+1H), 169.1 (M+1H), 239.2 (M+1H), 267.3 (M+1H), 285.2 (M+1H), 371.4 (M+1H) and 415.2 (M+1H), and the maximum proportion (5.63%) substance of all degradation products (126.0 (M+1H)) is 40-100 times less toxic than MTX. A mechanism for the removal and degradation of mitoxantrone was proposed. Besides, actual water experiments confirmed that the maximum removal capacity of MTX by g-C3N4@Fe/Pd is up to 492.4 mg/g (0.02 g/L, 10 ppm).

Fabrication of a molecularly imprinted polymer-based electrochemical sensor for the selective assay of antipsychotic drug clozapine and performance comparison with LC-MS/MS.

Clozapine (CLO) is an atypical antipsychotic drug indicated for the treatment of schizophrenia. The treatment effectiveness of CLO is better than that of other atypical antipsychotics, and it has the advantage of being able to determine its effectiveness by measuring its concentration in the patient's blood. Thus, sensitive, selective, and accurate determination of CLO in blood is highly significant for treatment monitoring. This study describes the design and fabrication of a molecularly imprinted polymer (MIP)-based electrochemical sensor for CLO determination. This is the first MIP-based electrochemical application in the literature for CLO determination. Employing the thermal polymerization approach, the MIP was formed on the glassy carbon electrode (GCE) using CLO as the template, trans-3-(3-Pyridyl)acrylic acid (3,3-TA) as the functional monomer, and the support of zinc oxide nanoparticles (ZnO NPs). Elaborate characterizations in terms of surface morphology and electrochemistry were performed via scanning electron microscopy (SEM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) methods. An indirect approach was employed to determine CLO in standard solution, real human biological samples, and tablet formulation, using 5 × 10-3 M [Fe(CN)6]3-/4- solution as the redox probe. The limit of detection (LOD) values for the standard solution and serum sample were calculated as 2.9 × 10-11 M and 6.01 × 10-12 M, respectively. These values and recovery studies confirmed the sensor's sensitivity and feasibility. The measurements in the presence of similarly structured compounds (olanzapine and quetiapine fumarate) verified the sensor's superior selectivity. Moreover, the developed sensor's performance was compared and verified using an LC-MS/MS method using the student's t-test and F-test.

Investigation of the Cholesterol Biosynthesis by Heart-Cut Liquid Chromatography and Mass Spectrometric Detection.

The biosynthesis and homeostasis of cholesterol are essential for cellular function. Cholesterol is a major lipid with multiple roles in membrane stability, signaling, or as a precursor for other molecules. Because of the structural similarity of the sterols involved in the biosynthesis, their accurate identification and quantification is still challenging. Moreover, the huge difference in the concentration of cholesterol and its precursors can cause interferences during the detection. To overcome these problems, a heart-cut liquid chromatographic method was developed by evaluating 38 different columns to achieve optimal separation. The method efficiently separates all sterol biosynthesis intermediates, with detection limits in the low nmol/L-range and an upper limit of quantification of 9 mmol/L for cholesterol by using triple quadrupole mass spectrometric detection. Investigation of lung carcinoma cells treated with statins demonstrated the capability to detect a biological response, showing inhibition of sterol synthesis. This technique offers a robust tool for studying cholesterol biosynthesis and its role in disease.

ISO-upregulated BECN1 specifically promotes LC3B-dependent autophagy and anticancer activity in invasive bladder cancer.

Isorhapontigenin (ISO), an active compound isolated from the Chinese herb Gnetum Cleistostachyum, exhibited strong preventive and therapeutic effects on bladder cancer (BC) both in vitro and in vivo. Our previous studies revealed that ISO-induced autophagy is crucial for its anti-cancer activity. However, the underlying mechanism remains unclear. Here, we showed that BECN1, an important autophagic protein, was induced by ISO treatment and played crucial roles in ISO-induced late phase of LC3B-dependent, and LC3A-independent autophagy, as well as anti-cancer activity. Downregulation of BECN1 was observed in human BCs and BBN-induced mouse invasive BC tissues, whereas co-treatment with ISO completely reversed BECN1 downregulation in BBN-induced mouse invasive BCs. Consistently, ISO treatment significantly increased BECN1 expression in vitro in a dose- and time-dependent manner. Depletion of BECN1 significantly impaired LC3B-dependent autophagy following ISO treatment, as well as abolished the inhibitory effect of ISO on anchorage-independent growth of human BC cells. Mechanistic studies revealed that BECN1 induction was mediated by ISO downregulation of c-Myc, which resulted in miR-613 reduction, in turn leading to increased NCL translation and further promoting NCL binding to BECN1 mRNA, subsequently stabilizing BECN1 mRNA. In conclusion, our results demonstrate that by activating c-Myc/miR-613/NCL axis, ISO treatment results in BECN1 posttranscriptional upregulation, which specifically initiates LC3B-dependent autophagy and anti-cancer activity. Our findings further strengths our application of ISO for therapy of high-grade invasive BC (HGIBC) patients.

Challenges and Perspectives on the Adoption of Cystatin C testing in China: A laboratory technician's perspective.

Cystatin C (CysC) belongs to the cysteine protease inhibitor superfamily and is produced by all nucleated cells in the body in very stable amounts independent of age, sex, diet, and muscle mass. CysC is considered an ideal biomarker for assessing glomerular filtration rate (GFR) compared to traditional biomarkers for assessing GFR, such as creatinine. However, CysC is not sufficiently utilized for GFR assessment by clinicians, probably for various reasons such as insufficient understanding among clinicians or a lack of standardized quantitative methods. This review discusses and analyzes the aforementioned issues from the perspective of laboratory technicians.

Phytochemical analysis and wound healing properties of Malva parviflora L. ethanolic extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Scientific publications documented the use of plants from Genus Malva to treat inflammatory diseases and skin disorders by our ancestors. Malva parviflora L. has reported benefits for wound healing in traditional medicine; however, there is a lack of experimental study to validate these claims. AIM: We initiated this study to explore the metabolites and verify the wound healing properties of M. parviflora using in vivo and in vitro models. MATERIALS AND METHODS: Liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was used to identify the ethanolic extract different metabolites. Additionally, total phenolic content was determined using Folin-Ciocalteu reagent. To verify the extract wound healing potential, an in vivo rat wound excision model was employed. Round wounds (5 mm in diameter) were created by a sterile biopsy punch needle. The wounds were treated with plant extracts (2.5% and 5%) as well as a commercially available wound healing product (Mebo®) for 10 days. The results were assessed as follows: 1) Measuring the reduction% in wound area compared to the original wound size. 2) Evaluation of the levels of wound healing biomarkers, namely collagen type I (Col-1), alpha smooth muscle actin (α-SMA), extracellular signal-regulated kinases-1 (ERK1), and matrix metalloproteinase-9 (MMP9) levels. 3) Performing histopathological examination of the wound tissue. The antioxidant properties of the M. parviflora leaves ethanolic extract were investigated using various assays: total antioxidant capacity (TAC), iron reducing power (IRP), 1,1-Diphenyl-2-picryl-hydrazyl (DPPH), and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals scavenging assays. Furthermore, the anti-inflammatory activity was confirmed by calculating the inhibition percentages of protein denaturation and the activity of the proteinase enzyme. RESULTS: Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis revealed the presence of various secondary metabolites in M. parviflora ethanolic extract, including phenolic acids (cinnamic and ferulic acids), flavonoids (quercetin and "iso"rhamnetin monoglucuronides), fatty acids (hydroxy-octadecatrienoic and oxo-octadecatrienoic acids), in addition to chlorophyll derivatives and carotenoids (pheophorbide-a and lutein, respectively). Malva extracts significantly reduced wound size compared to untreated control group. The extracts also promoted wound healing by upregulating collagen I, α-SMA, and ERK1 levels, while downregulating MMP9 expression. Notably, the effect of 2.5% and 5% extracts was similar or exceeds those of Mebo®, supported by histopathological results. Finally, M. parviflora ethanolic extract exhibited antioxidant and anti-inflammatory potentials comparable to the used standards. CONCLUSION: Our study provides evidence-based support for the wound healing properties of M. parviflora L. leaves ethanolic extract. This is further strengthened by the fact that many of the identified metabolites possess wound healing, antioxidant, and /or anti-inflammatory activities.

Azaspiracid-59 accumulation and transformation in mussels (Mytilus edulis) after feeding with Azadinium poporum (Dinophyceae).

Azaspiracid-59 (AZA-59) was detected in plankton in coastal waters of the Pacific Northwest USA. Given that bivalves metabolize and transform accumulated phycotoxins, a strain of Azadinium poporum isolated from the coast of Washington State that is a known producer of AZA-59 was used in a controlled feeding experiment with mussels (Mytilus edulis) to assess AZA-59 accumulation rates and transformation into shellfish metabolites. Mussels started feeding immediately after the addition of A. poporum. Mussels were generally healthy during the entire experimental exposure of 18 days with prevailingly high rates of clearance (ca 100 mL per mussel and hour) and ingestion. Mussel were extracted after different exposure times and were analyzed by liquid chromatography coupled with low- and high-resolution mass spectrometry. In the course of the experiment a number of putative AZA-59 metabolites were detected including hydroxyl and carboxy analogs that corresponded with previously reported mussel metabolites of AZA-1. A significant formation of 3-OH fatty acid acyl esters relative to free AZAs was observed through the time course of the study, with numerous fatty acid ester variants of AZA-59 confirmed. These results illustrate the potential for metabolism of AZA59 in shellfish and provide important information for local AZA monitoring and toxicity testing along the Northern Pacific US coast.