Liquid Chromatography-electrospray Mass Spectrometry Research Articles

Efficacy of Veronica incana for Treating Osteoarthritis Induced by Monosodium Iodoacetate in Rats.

The aim of this study is to investigate the efficacy and the underlying mechanism of Veronica incana in osteoarthritis (OA) induced by intraarticular injection of monosodium iodoacetate (MIA). The selected major four compounds (A-D) of V. incana were found from fractions 3 and 4. Its structure elucidation was determined by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) data analysis and nuclear magnetic resonance (NMR) data comparison with literature. MIA (50 μL with 80 mg/mL) for the animal experiment was injected into the right knee joint. The V. incana was administered orally every day to rats for 14 days from 7 days after MIA treatment. Finally, we confirmed the four compounds: (A) verproside; (B) catalposide; (C) 6-vanilloylcatapol; and (D) 6-isovanilloylcatapol. When we evaluated the effect of V. incana on the MIA injection-induced knee OA model, there were a noticeable initial decreased in hind paw weight-bearing distribution compared to the Normal group (P < .001), but V. incana supplementation resulted in a significant increase in the weight-bearing distribution to the treated knee (P < .001). Moreover, the V. incana treatment led to a decrease in the levels of liver function enzymes and tissue malondialdehyde (P < .05 and .01). The V. incana significantly suppressed the inflammatory factors through the nuclear factor-kappa B signaling pathway and downregulated the expression of matrix metalloproteinases, which are involved in the degradation of the extracellular matrix (P < .01 and .001). In addition, we confirmed the alleviation of cartilage degeneration through tissue stains. In conclusion, this study confirmed the major four compounds of V. incana and suggested that V. incana could serve as an anti-inflammatory candidate agent for patients with OA.

Estimation of dihydroartemisinin in human plasma using a highly sensitive LTQ Orbitrap mass spectrometer with Xcalibur software.

Background: Artemether (ARM), the O-methyl ether prodrug of dihydroartemisinin (DHA), is considered a first-line antimalarial agent. Artemether is extensively metabolized in vivo to its active metabolite DHA, and therefore its determination offers considerable difficulties. In the present study, DHA identification and estimation were accurately performed by the mass spectrometric analysis, using a high-resolution liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) LTQ Orbitrap hybrid mass spectrometer. Methods: The plasma samples were taken from healthy volunteers, and the spiked plasma was extracted by adding 1mL of a mixture of dichloromethane and tert.-methyl butyl ether (8:2v/v) to 0.5mL of plasma. The internal standard solution (artemisinin 500ng/mL) was added to the plasma samples. After vertexing and centrifugation, the organic layer was separated and transferred into another tube and dried under nitrogen. The residue was reconstituted in 100μL of acetonitrile and was injected onto the LC-MS system for analysis. Measurement of standards and samples was carried out isocratically on a Surveyor HPLC system combined with an LTQ Orbitrap mass spectrometer using an ACE 5 C18-PFP column. Mobile phase A consisted of 0.1% v/v formic acid in water, Mobile phase B consisted of acetonitrile only, and isocratic elution was carried out with A:B 20:80, v/v. The flow rate was 500μL/min. The ESI interface was operated in a positive ion mode with a spray voltage of 4.5kV. Results: Artemether is not a very biologically stable compound and is immediately metabolized to its active metabolite dihydroartemisinin, so no clear peak was observed for artemether. Both artemether and DHA after ionization undergo neutral losses of methanol and water, respectively, in the source of the mass spectrometer. The ions observed were (MH-H2O) m/z 267.15 for DHA and (MH-m/z 283.15 for internal standard artemisinin. The method was validated according to international guidelines. Discussion: The validated method was applied successfully for the determination and quantification of DHA in plasma samples. This method works well for the extraction of drugs, and the Orbitrap system with the help of Xcalibur software accurately and precisely determines the concentration of DHA in spiked as well as volunteer's plasma.

Polybutylene terephthalate-based stationary phase for ion-pair-free reversed-phase liquid chromatography of small interfering RNA. Part 2: Use for selective comprehensive two-dimensional liquid chromatography

With the increasing numbers of nucleic acid-based pharmaceuticals like antisense oligonucleotides (ASO), small interfering ribonucleic acid (siRNA) entering the market, research facilities, pharmaceutical industries and also regulatory authorities have been looking for efficient analytical methods for these synthetic oligonucleotides (ON). Besides of conventional one-dimensional (1D) reversed-phase liquid chromatography with or without ion-pairing (IP-RP-LC, RP-LC), hydrophilic liquid chromatography (HILIC) and mixed-mode chromatography (MMC), two-dimensional (2D) approaches combining two orthogonal chromatographic techniques also become more relevant due to the high structural complexity of oligonucleotides. Recently, we tested a polybutylene terephthalate(PBT)-based stationary phase under ion-pairing free RP mode for the liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) analysis of siRNA (Patisiran). In this study, retention profile and chromatographic orthogonality, respectively, were compared to other LC-modes like HILIC, IP-RPLC, another ion-pair free cholesterol-bonded RPLC and MMC considering their normalized retention times. Finally, because of higher orthogonality, the ion-pairing free PBT-bonded RPLC as first dimension (1D) was hyphenated with HILIC in the second dimension (2D) in a selective comprehensive 2D-LC setup leading to an enhanced resolution for peak purity evaluation of the main ON entities.

Headspace GC/MS and LC/MS analysis of bioactive compounds from Gossypium barbadense L. stem and assessment of their antimicrobial and cytotoxic activities

ABSTRACT. In line with the global interest and demand in finding sustainable ways to the emerging antimicrobial and cytotoxic activities, our study highlights the unique chemical composition of G. barbadense stem. Petroleum ether extracts were analyzed via headspace-gas chromatography/mass spectrometry (HS-GC/MS) revealing 48 compounds, thirteen major and accounting for 56.29% of total peak areas. By using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) identify 16 major phenolic compounds from the methanolic extract. Both petroleum ether and methanolic extracts were tested for antibacterial activity against four pathogenic bacterial strains (Escherichia coli ATCC10536, Staphylococcus aureus ATCC6538, Salmonella enterica ATCC 14028, and Pseudomonas aeruginosa ATCC 9027) by using disk diffusion method. The antifungal activity against two fungal strains (Aspergillus niger and Aspergillus fumigatus) was evaluated. The results were compared to the activity of ciprofloxacin as a stander. The in-vitro cytotoxic activity of the methanolic extract showed a potent effect against liver (HEPG2) with IC50 values (18.8±7.4 μg/mL) and moderate activity against colon (HCT116) and breast (MCF7) cancer.&#x0D; &#x0D; KEY WORDS: Gossypium Barbadense L. Stem, Headspace/GC/MS, LC-ESI-MS, Bioactive compounds, Antimicrobial, Cytotoxic activities&#x0D; &#x0D; Bull. Chem. Soc. Ethiop. 2023, 37(4), 1021-1032. &#x0D; DOI: https://dx.doi.org/10.4314/bcse.v37i4.17

Identification of metabolic biomarkers for diagnosis of epithelial ovarian cancer using internal extraction electrospray ionization mass spectrometry (iEESI-MS).

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancies. The poor prognosis of EOC is mainly due to its asymptomatic early stage, lack of effective screening methods, and a late diagnosis in the advanced stages of the disease. This study investigated metabolomic abnormalities in epithelial ovarian cancers. Our study developed a novel strategy to rapidly identify the metabolic biomarkers in the plasma of the EOC patients using Internal Extraction Electrospray Ionization Mass Spectrometry (IEESI-MS) and Liquid Chromatography-mass Spectrometry (HPLC-MS), which could distinguish the differential metabolites in between plasma samples collected from 98 patients with epithelial ovarian cancer, including 78 cases with original (P), and 20 cases with self-configuration (ZP), as well as 60 healthy subjects, including 30 cases in the original sample (H), 30 cases in self-configuration (ZH), and 6 cases in a blind sample (B). Our study detected 880 metabolites based on criteria variable importance in projection (VIP) > 1, among which 26 metabolites were selected for further identification. They are mainly metabolism-related lipids, amino acids, nucleic acids, and others. The metabolic pathways associated with the differential metabolites were explored by the KEGG analysis, a comprehensive database that integrates genome, chemistry, and system function information. The abnormal metabolites of EOC patients identified by IEESI-MS and HPLC-MS included Lysophosphatidylcholine (16:0) [Lyso PC (16:0)], L-Phenylalanine, L-Leucine, Phenylpyruvic acid, L-Tryptophan, and L-Histidine. Identifying the abnormal metabolites of EOC patients through metabolomics analyses could provide a new strategy to identify valuable potential biomarkers for the screening and early diagnosis of EOC.

Widespread occurrence of in-source fragmentation in the analysis of natural compounds by LC-ESI-MS.

The In-Source Fragmentation (ISF) of analyte or co-eluting substances produces unintentional fragment ions, which hampers the identification and quantification by liquid chromatography-mass spectrometry (LC-MS). Natural compounds derived from plants also contain fragile moieties that may undergo ISF. However, the characteristics of ISF of natural compounds in LC-MS are still unclear. The ISF behavior of 214 natural compounds was assayed in liquid chromatography-Q/orbitrap mass spectrometry in electrospray ionization mode (ESI) and the extent of ISF was evaluated. Up to 82% of tested compounds undergo ISF and half of the tested natural compounds that contain more than one fragile moiety underwent successive and severe ISF to generate serial structurally-related ISF products. The major ISF-altering moieties for natural compounds were hydroxyl, lactone, glycosyl, and ether, resulting in neutral loss of H2 O or CO, deglycosylation, or cleavage of ether bond, respectively. Some compounds such as terpenoids undergo severe ISF and less than 1% parent form can be observed. For natural compounds, the ISF product with similar structures more likely to cause interference in analysis because the ISF products may share identical mass-to-charge ratio and similar MS2 fragmentation patterns with precursor ions of the homologs in plants. Furthermore, the severe ISF may cause a false negative in the identification of the parent form. In summary, ISF was a highly frequent phenomenon for analysis of natural compounds by LC-ESI-MS, extensive and successive ISF of natural products may cause misannotation and misidentification with the homologs in plants. It should raise awareness of ISF interference during the analysis of natural compounds.

Anti-Plasmodial Activity and Mass Spectrometric Profiling of Peptide-enriched Methanol/Water Extract of Leaves from the Himalayan Viola canescens Wall

Background: Plants belonging to the genus Viola and family Violaceae are well known in Ayurvedic and Unani medicine for their pharmaceutical importance. Viola, commonly known as Banafsha, has been traditionally used as an anti-malarial. The goal of this study was to determine the anti-Plasmodial activity of the cyclotide-rich crude methanol/water extract of leaves from Viola canescens Wall and to analyze its constituents by mass spectrometry (MS). Materials and Methods: Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) involving quadrupole time-of-flight (Q-ToF) and matrix-assisted laser desorption/ionization (MALDI)-time-of-flight-mass spectrometry (ToF-MS) were applied to investigate the cyclotides rich in methanol/water extract. The intact molecular masses were observed in the range of 2,800–3,400 Daltons, suggesting the presence of peptides. Since various head-to-tail cyclized and intramolecularly disulfide-bonded peptides known as cyclotides have been identified in the Violaceae family, we wanted to verify whether cyclotides are present in our extract. So, the intact masses inferred from the observed spectra were interrogated with Cybase ( www.cybase.org.au ), which is a database on plant cyclotides. Results: We observed promising anti-Plasmodial activity (IC50 44 µg/ml) in the cyclotide-rich methanol/water extract prepared from the leaves of the Himalayan V. canescens Wall. The output obtained from Cybase confirmed the presence of about 20 different cyclotides in our extracts. However, no sequence information could be obtained from Cybase for 14 intact molecular masses, which were inferred from the observed LC-ESI-MS data. Hence, these could correspond to novel peptide/cyclotide sequences, which might be characteristic of Himalayan V. canescens. Conclusion: This is the first report on the anti-Plasmodial activity of the cyclotide-enriched methanol/water crude extract of the leaves of V. canescens.

Is the Novel Slot Blot a Useful Method for Quantification of Intracellular Advanced Glycation End-Products?

Various types of advanced glycation end-products (AGEs) have been identified and studied. I have reported a novel slot blot analysis to quantify two types of AGEs, glyceraldehyde-derived AGEs, also called toxic AGEs (TAGE), and 1,5-anhydro-D-fructose AGEs. The traditional slot blot method has been used for the detection and quantification of RNA, DNA, and proteins since around 1980 and is one of the more commonly used analog technologies to date. However, the novel slot blot analysis has been used to quantify AGEs from 2017 to 2022. Its characteristics include (i) use of a lysis buffer containing tris-(hydroxymethyl)-aminomethane, urea, thiourea, and 3-[3-(cholamidopropyl)-dimetyl-ammonio]-1-propane sulfonate (a lysis buffer with a composition similar to that used in two-dimensional gel electrophoresis-based proteomics analysis); (ii) probing of AGE-modified bovine serum albumin (e.g., standard AGE aliquots); and (iii) use of polyvinylidene difluoride membranes. In this review, the previously used quantification methods of slot blot, western blot, immunostaining, enzyme-linked immunosorbent assay, gas chromatography-mass spectrometry (MS), matrix-associated laser desorption/ionization-MS, and liquid chromatography-electrospray ionization-MS are described. Lastly, the advantages and disadvantages of the novel slot blot compared to the above methods are discussed.

Continuous Electrospray Ionization Mass Spectrometry Assay for Measuring Phospholipase Activity against Liposomes.

Phospholipases have diverse roles in lipid and cell membrane biology. In animal venoms, they can have roles as neurotoxins or myotoxins that disrupt the integrity of cell membranes. In this work, we describe a temperature-controlled, continuous electrospray ionization mass spectrometry (ESI-MS) assay for measuring phospholipase A2 activity against liposomes. The enzyme used in this assay was paradoxin, which is a neurotoxic trimeric phospholipase A2 from inland taipan snake venom. Previously developed ESI-MS-based phospholipase assays have been discontinuous and analyzed hydrolysis of single lipid molecules by liquid chromatography ESI-MS. In this work, a continuous assay was developed against liposomes, a more complex substrate that more closely reflects the natural substrate for paradoxin. The assay confirmed the requirement for Ca2+ and allowed measurement of Michaelis-Menten-type parameters. The use of ESI-MS for lipid detection enabled nuanced insights into the effect of changing assay conditions not only on the enzyme but also on the liposome substrate. Changing the metal ion concentrations did not significantly change the liposomes but did affect enzymatic activity. Increasing temperature did not substantially affect the secondary structure of paradoxin but affected liposome size, resulting in increased enzymatic activity consistent with the disruption of the phosphatidylcholine membrane, increasing accessibility of sn-2 ester bonds. The continuous ESI-MS method described herein can be applied to other enzyme reactions, particularly those which utilize complex lipid substrates.

Biological Activity of Cyclic Peptide Extracted from Sphaeranthus amaranthoides Using De Novo Sequencing Strategy by Mass Spectrometry for Cancer.

Though there are several advancements and developments in cancer therapy, the treatment remains challenging. In recent years, the antimicrobial peptides (AMPs) from traditional herbs are focused for identifying and developing potential anticancer molecules. In this study, AMPs are identified from Sphaeranthus amaranthoides, a natural medicinal herb widely used as a crucial immune stimulant in Indian medicine. A total of 86 peptide traces were identified using liquid-chromatography-electrospray-ionisation mass spectrometry (LC-ESI-MS). Among them, three peptides were sequenced using the manual de novo sequencing technique. The in-silico prediction revealed that SA923 is a cyclic peptide with C-N terminal interaction of the carbon atom of ASP7 with the nitrogen atom of GLU1 (1ELVFYRD7). Thus, SA923 is presented under the orbitides class of peptides, which lack the disulfide bonds for cyclization. In addition, SA923, steered with the physicochemical properties and support vector machine (SVM) algorithm mentioned for the segment, has the highest in silico anticancer potential. Further, the in vitro cytotoxicity assay revealed the peptide has anti-proliferative activity, and toxicity studies were demonstrated in Danio rerio (zebrafish) embryos.

Resonance Raman and Visible Micro-Spectroscopy for the In-Vivo and In-Vitro Characterization of Anthocyanin-Based Pigments in Blue and Violet Flowers: A Comparison with HPLC-ESI- MS Analysis of the Extracts

Microanalysis techniques based on resonance Raman and reflection visible spectroscopy have been applied to the characterization of pigments responsible for the blue or violet coloration in flowers; in particular of Lobelia erinus, Campanula portenschlagiana, Cineraria, Viola tricolor, Anemone coronaria, Agapanthus, Platycodon, Salvia farinacea, Plumbago capensis, Ceratostigma plumbaginoides, Commelina communis and Salvia patens. The spectroscopic methods were applied both in vivo on the flower petals and in vitro on extracts obtained through a procedure based on SPE (solid-phase extraction) optimized for minimal quantities of vegetable raw material. Different patterns obtained for the Raman spectra have been correlated, also on the basis of density functional theory (DFT) calculations, with different schemes of substitution of the benzopyrilium nucleus of the anthocyanins and with various possible forms of copigmentation responsible for the stabilization of the blue color. The results obtained were verified by comparison with the analysis of the extracts by HPLC-ESI-MS (liquid chromatography-electrospray ionization-mass spectrometry).

Antimicrobial and Antiviral Compounds of Phlomis viscosa Poiret

Phlomis viscosa Poiret (an evergreen shrub) represents a valuable source of medicinal compounds. In this study, we discovered compounds with antimicrobial and antiviral properties. The aim of this study was to identify compounds of P. viscosa and estimate the antimicrobial and antiviral activity of its phytochemicals. The volatile compounds were identified using gas chromatography/mass spectrometry (GC/MS) analysis. For the identification of nonvolatile components of the extracts, high-performance liquid chromatography (HPLC), liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) were applied. Quercetin 3-O-rutinoside and hesperidin caused a significant decrease in the bacterial concentration of Agrobacterium tumefaciens, Xylella fastidiosa and Pseudomonas syringae (p < 0.001). The growth of drug-resistant microorganisms (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Serratia marcescens and Salmonella enteritidis) was inhibited by quercetin 3-O-rutinoside, quercetin 3-O-arabinoside and hesperidin. In addition, these compounds demonstrated antiquorum-sensing properties. Diosmin, hesperidin and quercetin 3-O-arabinoside significantly inhibited varicella zoster virus (VZV) (p < 0.001). Quercetin 3-O-rutinoside and quercetin 3-O-arabinoside were effective against herpes simplex virus 1 (HSV-1), including mutant strains.

Hydrophilic interaction liquid chromatography–electrospray ionization mass spectrometry combined with fabric phase sorptive extraction for therapeutic drug monitoring of pioglitazone, repaglinide, and nateglinide in human plasma

Polypharmacy in type 2 diabetes is an issue of major concern as the prescription of multiple medi-cations for the management of diabetes-associated comorbidities can lead to drug-to-drug interactions, which can pose serious risks to patients’ health. Within this context, the development of bioanalytical methods for monitoring the therapeutic levels of antidiabetic drugs is notably useful to ensure patients’ safety. In the present work, a liquid chromatography–mass spectrometry method for the quantitation of pioglitazone, repaglinide, and nateglinide in human plasma is described. Sample preparation was performed by fabric phase sorptive extraction (FPSE), and hydrophilic interaction liquid chromatography (HILIC) was implemented for the chromatographic separation of the analytes, using a ZIC®-cHILIC analytical column (150 × 2.1 mm, 3 µm) under isocratic elution. The mobile phase consisted of 10 mM ammonium formate aqueous solution (pH = 6.5)/ acetonitrile, 10/90 v/v, and was pumped at a flow rate of 0.2 mL min−1. Design of Experiments was used during the development of the sample preparation method to gain deeper insight into the effect of various experimental parameters on extraction efficiency, their potential interactions and to optimize the recovery rates of the analytes. The linearity of the assay was assessed over the ranges of 25 to 2000, 6.25 to 500, and 125 to 10000 ng mL−1 for pioglitazone, repaglinide, and nateglinide, respectively. The presented method was fully validated and can be used for the therapeutic monitoring of the targeted analytes in human plasma samples.

Phytochemical Screening, Antioxidant Potential, and LC-ESI-MS Profiling of Ephedra alata and Ephedra altissima Seeds Naturally Growing in Tunisia.

Most Ephedra species are adapted to arid and desert conditions and are widely used in folk medicine to treat several disorders. The design of the current study was to determine the functional properties of seeds of two Ephedra species (E. alata and E. altissima) naturally growing in Tunisian arid zones by evaluating their mineral contents and bioactive compounds. The flame atomic absorption spectrometry revealed that seeds contained remarkable amounts of sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), copper (Cu), zinc (Zn), and iron (Fe). The colorimetric investigation revealed high total polyphenol, flavonoid, and condensed tannin contents. Furthermore, by utilizing high-performance liquid chromatography-electrospray ionization-mass spectrometry method (HPLC-ESI/MS), a total of 11 phenolics were identified and quantified including 7 flavonoid compounds and 4 phenolic acids that were mostly predominated by gallic acid and quercetrin. Results so far have been very encouraging and proved that Ephedra seeds are a valuable source of natural bioactive compounds and minerals which could potentially be used for industrial and pharmaceutical purposes.

Ionic Liquid-Dispersive Micro-Extraction and Detection by High Performance Liquid Chromatography-Mass Spectrometry for Antifouling Biocides in Water.

A simple analytical method was developed and evaluated for the determination of two antifouling biocides using an ionic liquid-dispersive liquid-liquid micro-extraction (IL-DLLME) and a high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) analysis. Irgarol 1051 and Sea-Nine 211 were extracted from deionized water, lake water, and seawater using IL 1-hexyl-3-methylimidazolium hexafluorophosphate ([HMIm][PF6]) and ethyl acetate as the extraction solvent and the dispersion solvent. Several factors were considered, including the type and volume of extraction and dispersive solvent, IL amount, sample pH, salt effect, and cooling temperature. The developed method resulted in a recovery range of 78.7-90.3%, with a relative standard deviation (RSD, n = 3) less than 7.5%. The analytes were enriched greater than 40-fold, and the limits of detection (LOD) for two antifouling biocides were 0.01-0.1 μg L-1. The method was effectively applied for the analysis of real samples of freshwater as well as samples of seawater.

Characterization and Comparison of Steroidal Glycosides from Polygonatum Species by High-Performance Liquid Chromatography–Electrospray Ionization Mass Spectrometry

Polygonatum species have been used as traditional medicines and functional foods in Asia and Europe since ancient times. In this study, a fast and simple method based on liquid chromatography coupled with electrospray ionization mass spectrometry (LC-ESI-MS) was developed to systematically analyze and identify the steroidal glycosides in four major Polygonatum species distributed in Japan, including P. odoratum, P. falcatum, P. macranthum, and P. sibiricum. As a result, 31 steroidal glycosides were tentatively identified, including 18 known and 13 previously unreported glycosides. Their structures were identified by the interpretation of chromatographic behavior and ESI-MS fragmentation patterns. The identification of 31 steroidal glycosides was indicative of a common biogenetic pathway in Polygonatum species. Our study disclosed the chemical profiling of steroidal glycosides in the plants of Polygonatum species, which will benefit better phytochemotaxonomical and phytochemical understanding and quality control for their medicinal usage.

Mass Spectrometry of Neutral Glycosphingolipids.

This chapter describes the protocols for mass spectrometry (MS) applied to the structural characterization of neutral glycosphingolipids (GSLs) and the determination of neutral GSL contents in biological materials. The structural characterization is performed by thin layer chromatography-matrix assisted laser desorption ionization/mass spectrometry (TLC-MALDI/MS) and liquid chromatography-electrospray ionization/mass spectrometry (LC-ESI/MS) with reversed phase separation. The content determination is carried out by LC-ESI/MS with multiple reaction monitoring (MRM). These protocols provide clues for the functions of neutral GSLs at the level of a single GSL molecular species.

Characterization and anticancer potential of Withania somnifera fruit bioactives (a native species to Pakistan) using gas chromatography-mass spectrometer, nuclear magnetic resonance and liquid chromatography-mass spectrometry-electrospray ionization.

Withania somnifera (W. somnifera) is a plant with remarkable pharmacological properties. The plant has an impressive profile of medicinal uses in the folk medicine system of several civilizations. This comprehensive study is aimed to characterize phytochemicals in fruit of W. somnifera and tested for anticancer potential to find out active candidate in disease prevention and treatment. The bioactive components from W. somn-ifera fruit were extracted with polar and non-polar solvents. Anticancer potential of the isolated bioactive was assessed against different cancer cell lines through MTT assay and Incucytes imaging analysis. The extracts were characterized for secondary metabolites using GC-MS (gas chromatography-mass spectrometer), LCMS (liquid chromatography-mass spectrometry)-ESI (electrospray Ionization) and 1H-NMR (electrospray Ionization) techniques. Both freeze-dried and rotary evaporator con-densed extracts exhibited anticancer potential against MDA-MB-231, MCF7- SKOV3 and SKBR3 cell lines. The tested extracts have cell growth inhibition potential ag-ainst mammalian cancer cell line. Hexacosanedioic acid purified from -hexane extract through HPLC was inves-tigated for its cytotoxicity against breast cancer cell line SKBR3 by using Incucytes imaging analysis. We found that a variety of bioactive compounds existed in this plant. One identified compound that was not investigated for cytotoxicity in previous studies was purified and its application showed cytotoxicity on breast cancer cell lines. A number of bioactive identified from fruit may have an effective potential for development into chemotherapy drugs.

Bivalves Transmissible Neoplasia: Biochemical Aspects of Contagious Cancer in a Clam Macoma Balthica.

Occurring in marine invertebrates infectious haemic neoplasia (bivalves transmissible neoplasia, BTN) arises from genome instabilities leading to multilevel malfunctions and unregulated cell division of presumably haemocyte precursors. As its biochemical characterisation remains unknown, we here present the first data describing selected aspects of the physiology and biochemistry of the disease a in model clam Macoma balthica. We chose free amino acids (FAA) composition, mitochondrial respiration and enzymatic activity, oxidative stress enzymes activities and corticosteroids profile as markers of this contagious cancer. Selected markers were measured in neoplastic and healthy clams and two tissue types, haemolymph and solid tissue. FAA composition was assessed in the haemolymph samples using high performance liquid chromatography-mass spectrometry (LC/MS). Mitochondrial respiration analysis was performed on haemocytes using oxygen electrodes integrated system Seahorse XFp. Mitochondrial enzymes activities were measured using spectrophotometry (cytochrome oxidase, COX) and commercial kit (succinate dehydrogenase, SDH). Total Antioxidant Capacity (TAC), Acetylocholinesterase (AChE), Protein Carbonyl Content (CBO) and Malondialdehyde (MDA) levels were measured in the solid tissue using analytical kits, and glutathione (GSH) was measured spectrophotometrically. Corticosteroids profile, measured in the solid tissue, was obtained with Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (HPLC-ESI-MS/MS) technique. In both clam groups nine FAAs were detected with Asp, Glu, Pro, Ser constituting over 90% of total FAA content. Significantly higher Gln level was detected in BTN positive clams. In neoplastic clams, an impairment of mitochondrial metabolism was observed as a decrease in mitochondrial oxygen consumption and lower cytochrome c oxidase activity. In the neoplastic clams significantly higher concentration of low molecular weight antioxidants was found. Finally, we report high level of corticosterone and lower levels of dehydrocorticosterone, cortisol and cortisone in healthy clams and elevated cortisol level in BTN individuals. Neoplastic clams are characterized by altered mitochondrial metabolism, with a potential key role of glutamine (Gln) in cancer cells energy production. Despite low aerobic respiration, BTN cells have efficient antioxidative response to elevated concentration of ROS. Elevated cortisol level in BTN-positive clams may indicate an important role of this corticosteroid in cancer biochemistry. Thus, we here provide the first results of selected physiological and biochemical aspects of BTN, making an important step in studying cancer epidemiology in wildlife.

Structure analysis and tyrosinase, melanogenesis, α-glucosidase, and nonenzymatic glycation inhibitory activities of polymeric proanthocyanidins from the pulp of Crataegus pinnatifida Bge.

Crataegus pinnatifida Bge. is a worthwhile industrial crop for its extensive application in Chinese herbal pharmaceuticals, food, and other industries. In this study, proanthocyanidins were extracted from the pulp of Crataegus pinnatifida Bge., and the structure, antityrosinase, antimelanogenesis, anti-α-glucosidase, and anti-glycation activities of these compounds were systematically elucidated. The combined utilization of high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS), matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), and nuclear magnetic resonance (NMR) demonstrated that the proanthocyanidins were B-type procyanidin polymers consisting of epicatechin . These polymers were efficient, reversible, and competitive inhibitors of both tyrosinase and α-glucosidase with IC 50 values of 0.10 ± 0.01 mg/mL (for monophenolase), 0.16 ± 0.02 mg/mL (for diphenolase), and 0.50 ± 0.02 µg/mL, respectively. Moreover, the process of nonenzymatic glycation was also delayed by proanthocyanidins. Further cell assays suggested that these polymers strongly inhibited intracellular tyrosinase activity and melanogenesis and induced apoptosis in mouse melanoma cells. The results of circular dichroism (CD), molecular docking , and molecular dynamics (MD) consistently revealed that the inhibition of procyanidins on the two enzymes was primarily driven by hydrogen bonding and hydrophobic interactions, and the binding of epicatechin caused structural stretching and conformational modification of tyrosinase. Consequently, this study confirms novel tyrosinase, melanogenesis, α-glucosidase, and nonenzymatic glycation inhibitors, which might offer scientific evidence for potential applications in the cosmetics and pharmaceutical industries. • Proanthocyanidins can be produced industrially for a yield of 11.08%. • Various biological activities of proanthocyanidins enabled their wide applications. • Hydrogen bond and hydrophobic interaction loosened tyrosinase secondary structures. • Proanthocyanidins can reduce melanogenesis by down-regulating tyrosinase proteins.