Determination of ganglioside composition and structure in human brain hemangioma by chip-based nanoelectrospray ionization tandem mass spectrometry

We report here on a preliminary investigation of ganglioside composition and structure in human hemangioma, a benign tumor in the frontal cortex (HFC) in comparison to normal frontal cortex (NFC) tissue using for the first time advanced mass spectrometric methods based on fully automated chip-nanoelectrospray (nanoESI) high-capacity ion trap (HCT) and collision-induced dissociation (CID). The high ionization efficiency, sensitivity and reproducibility provided by the chip-nanoESI approach allowed for a reliable MS-based ganglioside comparative assay. Unlike NFC, ganglioside mixture extracted from HFC was found dominated by species of short glycan chains exhibiting lower overall sialic acid content. In HFC, only GT1 (d18:1/20:0), and GT3 (d18:1/25:1) polysialylated species were detected. Interestingly, none of these trisialylated forms was detected in NFC, suggesting that such components might selectively be associated with HFC. Unlike the case of previously investigated high malignancy gliosarcoma, in HFC one modified O-Ac-GD2 and one modified O-Ac-GM4 gangliosides were observed. This aspect suggests that these O-acetylated structures could be associated with cerebral tumors having reduced malignancy grade. Fragmentation analysis by CID in MS² mode using as precursors the ions corresponding to GT1 (d18:1/20:0) and GD1 (d18:1/20:0) provided data corroborating for the first time the presence of the common GT1a and GT1b isomers and the incidence of unusual GT1c and GT1d glycoforms in brain hemangioma tumor.      

Profiling and sequence analysis of gangliosides in human astrocytoma by high-resolution mass spectrometry

In this preliminary investigation, a low-grade astrocytoma (AcT) is investigated by high-resolution (HR) mass spectrometry (MS) aiming at characterization of gangliosides with potential biomarker value. The research was conducted towards a comparative mapping of ganglioside expression in AcT, its surrounding tissue (ST) and a normal control brain tissue (NT). HR MS was conducted in the negative ion mode nanoelectrospray ionization (nanoESI). Fragmentation analysis was carried out by collision-induced dissociation (CID) MS²–MS^4. Due to the high resolving power and mass accuracy, by comparative mapping of the ganglioside extracts from AcT, ST and NT, under identical conditions, 37 different species in AcT, 40 in ST and 56 in NT were identified. AcT and ST were found to contain 18 identical ganglioside components. Among all three specimens, ST extract presented the highest levels of sialylation, fucosylation and acetylation, a feature which might be correlated to the tumor expansion in the adjacent brain area. MS mapping indicated also that AcT, ST and NT share one doubly deprotonated molecule at m/z 1063.31, attributable to GT1(d18:1/18:0) or GT1(d18:0/18:1). CID MS²–MS⁴ on these particular ions detected in AcT and ST provided data supporting GT1c isomer in the investigated astrocytoma tissue. Our results show that HR MS has a remarkable potential in brain cancer research for the determination of tumor-associated markers and for their structural determination.

Gangliosidome of a Human Hippocampus in Temporal Lobe Epilepsy Resolved by High-Resolution Tandem Mass Spectrometry

In this study, we developed a high-resolution tandem mass spectrometry (HR MS) approach to assess presumed changes in gangliosidome of a human hippocampus affected by temporal lobe epilepsy (TLE) in comparison with a normal hippocampus. Gangliosides, membrane glycolipids, are particularly diverse and abundant in the human brain, and participate in ion transport and modulation of neuronal excitability. Changes in structural ganglioside pattern potentially linked to TLE molecular pathogenesis have not been explored in detail. Aiming to characterize TLE-specific gangliosidome, we analyzed the native gangliosides purified from a human hippocampal tissue sample affected by TLE and a control hippocampus using HR MS. Marked differences of ganglioside expression were shown in TLE vs. control, particularly with respect to the sialylation degree of components, discovered as a characteristic feature of TLE. Another major finding is the occurrence of tetrasialofucogangliosides in TLE and species modified by either O-acetylation or CH₃COO⁻. Structural analysis by higher-energy collisional dissociation (HCD) MS/MS gave rise to fragmentation patterns implying that the GQ1b (d18:1/18:0) isomer is specifically associated with TLE. Further investigation in a larger sample is needed in order to confirm the discovery of ganglioside structures specifically expressed in human TLE and to provide information on the probable role of gangliosides in the molecular events underlying seizures.     

Development and Validation of Stability-Indicating HPLC Methods for the Estimation of Lomefloxacin and Balofloxacin Oxidation Process under ACVA,H2O2, or KMnO4 Treatment. Kinetic Evaluation and Identification of Degradation Products by Mass Spectrometry

The oxidation of lomefloxacin (LOM) and balofloxacin (BAL) under the influence of azo initiator of radical reactions of 4,4′-azobis(4-cyanopentanoic acid) (ACVA) and H₂O₂ was examined. Oxidation using H₂O₂ was performed at room temperature while using ACVA at temperatures: 40, 50, 60 °C. Additionally, the oxidation process of BAL under the influence of KMnO₄ in an acidic medium was investigated. New stability-indicating HPLC methods were developed in order to evaluate the oxidation process. Chromatographic analysis was carried out using the Kinetex 5u XB—C18 100A column, Phenomenex (Torrance, CA, USA) (250 × 4.6 mm, 5 μm particle size, core shell type). The chromatographic separation was achieved while using isocratic elution and a mobile phase with the composition of 0.05 M phosphate buffer (pH = 3.20 adjusted with o-phosphoric acid) and acetonitrile (87:13 v/v for LOM; 80:20 v/v for BAL). The column was maintained at 30 °C. The methods were validated according to the ICH guidelines, and it was found that they met the acceptance criteria. An oxidation process followed kinetics of the second order reaction. The most probable structures of LOM and BAL degradation products formed were assigned by the UHPLC/MS/MS method.     

Highly sensitive localization analysis of gangliosides and sulfatides including structural isomers in mouse cerebellum sections by combination of laser microdissection and hydrophilic interaction liquid chromatography/electrospray ionization mass spectrometry with theoretically expanded multiple reaction monitoring

Liquid chromatography/electrospray ionization mass spectrometry (LC/ESI‐MS) is suitable for analysis of glycosphingolipids such as fragile gangliosides avoiding the use of the sialic acid elimination. However, it was not possible to distinguish the structural isomers such as GD1a and GD1b with reversed‐phase LC/ESI‐MS by hydrophobic interaction. Here we report an effective method for targeted analysis of theoretically expanded ganglioside molecular species including structural isomers by hydrophilic interaction liquid chromatography (HILIC)/ESI‐MS with multiple reaction monitoring (MRM). As a result of MRM analysis of glycosphingolipid mixtures from porcine brain, each of the lipid classes was detected within 25 min in the following order: sulfatides > GM3 > GM2 > GM1 > GD3 > GD1a > GD2 > GD1b > GT1a > GT1b > GQ1b. For the advanced application, localization analysis of postnatal day 15 (P15) mouse cerebellum layered structures was carried out by combination of MRM and laser microdissection (LMD). As a result, GM3, GD1a, GT1b and GQ1b were abundantly detected in the molecular and granular layers, whereas GM1 was widely presented in each layered structure. These gangliosides were mainly composed of d18:1‐18:0 and d18:1‐20:0, but GM3 was d18:1‐16:0 and d18:1‐20:0. Meanwhile, sulfatide molecular species were mostly localized in the myelinated fibers and scarcely found in the molecular layer. These results suggested that our method is suitable to detect a variety of ganglioside classes and sulfatides with high sensitivity at the molecular species level and effective for localization analysis of these glycosphingolipids from mouse brain sections. Copyright © 2010 John Wiley & Sons, Ltd.     

Assessment of the Molecular Expression and Structure of Gangliosides in Brain Metastasis of Lung Adenocarcinoma by an Advanced Approach Based on Fully Automated Chip-Nanoelectrospray Mass Spectrometry

Gangliosides (GGs), sialic acid-containing glycosphingolipids, are known to be involved in the invasive/metastatic behavior of brain tumor cells. Development of modern methods for determination of the variations in GG expression and structure during neoplastic cell transformation is a priority in the field of biomedical analysis. In this context, we report here on the first optimization and application of chip-based nanoelectrospray (NanoMate robot) mass spectrometry (MS) for the investigation of gangliosides in a secondary brain tumor. In our work a native GG mixture extracted and purified from brain metastasis of lung adenocarcinoma was screened by NanoMate robot coupled to a quadrupole time-of-flight MS. A native GG mixture from an age-matched healthy brain tissue, sampled and analyzed under identical conditions, served as a control. Comparative MS analysis demonstrated an evident dissimilarity in GG expression in the two tissue types. Brain metastasis is characterized by many species having a reduced N-acetylneuraminic acid (Neu5Ac) content, however, modified by fucosylation or O-acetylation such as Fuc-GM4, Fuc-GM3, di-O-Ac-GM1, O-Ac-GM3. In contrast, healthy brain tissue is dominated by longer structures exhibiting from mono- to hexasialylated sugar chains. Also, significant differences in ceramide composition were discovered. By tandem MS using collision-induced dissociation at low energies, brain metastasis-associated GD3 (d18:1/18:0) species as well as an uncommon Fuc-GM1 (d18:1/18:0) detected in the normal brain tissue could be structurally characterized. The novel protocol was able to provide a reliable compositional and structural characterization with high analysis pace and at a sensitivity situated in the fmol range.     

The Antimicrobial Activity of Origanum vulgare L. Correlated with the Gastrointestinal Perturbation in Patients with Metabolic Syndrome

(1) The metabolic syndrome (MS) promotes acute and chronic infections, due to the pro-inflammatory condition given by TNFα and IL6 or by affecting the microbiota. MS is also correlated with insulin resistance, causing inflammation and infections throughout the organism. (2) The purpose of this study was to track the effect of using the essential oil of Origanum vulgare L. (EOO) as an antibacterial treatment, compared to allopathic treatment with antibiotics in MS patients. A group of 106 people with MS was divided into four subgroups: L1—staphylococcal infection group, L2—Escherichia coli infection group, L3—streptococcal infection group with EOO treatment, and CG—control group without infection or treatment. (3) EOO is responsible for the antibacterial effect, and reduced minor uncomplicated infections. After a 10-day treatment, intestinal side effects were absent, improving the phase angle. (4) The results suggest that EOO may exhibit an antibacterial effect, similar to the antibiotic treatment, without promoting MS-specific dysbiosis, and it also improves the phase angle in patients, which is used as an index of health and cellular function.     

Evaluation of Antioxidant and Enzyme Inhibition Properties of Croton hirtus L’Hér. Extracts Obtained with Different Solvents

Croton hirtus L’Hér methanol extract was studied by NMR and two different LC-DAD-MSⁿ using electrospray (ESI) and atmospheric pressure chemical ionization (APCI) sources to obtain a quali-quantitative fingerprint. Forty different phytochemicals were identified, and twenty of them were quantified, whereas the main constituents were dihydro α ionol-O-[arabinosil(1-6) glucoside] (133 mg/g), dihydro β ionol-O-[arabinosil(1-6) glucoside] (80 mg/g), β-sitosterol (49 mg/g), and isorhamnetin-3-O-rutinoside (26 mg/g). C. hirtus was extracted with different solvents—namely, water, methanol, dichloromethane, and ethyl acetate—and the extracts were assayed using different in vitro tests. The methanolic extracts presented the highest 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS), and ferric reducing antioxidant power (FRAP) values. All the tested extracts exhibited inhibitory effects on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), with a higher activity observed for dichloromethane (AChE: 5.03 and BChE: 16.41 mgGALAE/g), while the methanolic extract showed highest impact against tyrosinase (49.83 mgKAE/g). Taken together, these findings suggest C. hirtus as a novel source of bioactive phytochemicals with potential for commercial development.     

Orbitrap mass spectrometry for monitoring the ganglioside pattern in human cerebellum development and aging

We have developed here a superior approach based on high‐resolution (HR) mass spectrometry (MS) for monitoring the changes occurring with development and aging in the composition and structure of cerebellar gangliosidome. The experiments were focused on the comparative screening and structural analysis of gangliosides expressed in fetal and aged cerebellum by Orbitrap MS with nanoelectrospray ionization (nanoESI) in the negative ion mode. The employed ultrahigh‐resolution MS platform allowed the discrimination, without the need of previous separation, of 159 ions corresponding to 120 distinct species in the native ganglioside mixtures from fetal and aged cerebellar biopsies, many more than detected before, when MS platforms of lower resolution were employed. A number of gangliosides, in particular polysialylated belonging to GT, GQ, GP, and GS classes, modified by O‐fucosylation, O‐acetylation, or CH₃COO^? were discovered here, for the first time in human cerebellum. These components, found differently expressed in fetal and aged tissues, indicated that the ganglioside profile in cerebellum is development stage‐ and age‐specific. Following the fragmentation analysis by high‐energy collision‐induced dissociation (HCD) tandem MS (MS/MS), we have also observed that the intimate structure of certain compounds has not changed during the development and aging of the brain, an aspect which could open new directions in the investigation of ganglioside biomarkers in cerebellar tissue.     

Chip-nanoelectrospray quadrupole time-of-flight tandem mass spectrometry of meningioma gangliosides: a preliminary study

A strategy combining high-performance thin layer chromatography (HPTLC), laser densitometry, and fully automated chip-based nanoelectrospray (nanoESIchip) performed on a NanoMate robot coupled to QTOF-MS was developed, optimized, and for the first time applied for mapping and structural identification of gangliosides (GGs) extracted and purified from a human angioblastic meningioma specimen. While HPTLC pattern indicated only seven fractions migrating as GM3, GM2, GM1, GD3, GD1a (nLD1, LD1), GD1b, GT1b, and possibly GD2, due to the high sensitivity, mass accuracy, and ability to ionize minor species in complex mixtures, nanoESIchip-QTOF MS was able to discover significantly more GG species than ever reported in meningioma. Thirty-four distinct glycosphingolipid components of which five asialo, one GM4, nine GM3, two GM2, two GD3, nine GM1, and six GD1 differing in their ceramide compositions were identified. All structures presented long-chain bases with 18 carbon atoms, while the length of the fatty acid was found to vary from C11 to C25. MS screening results indicated also that the diversity of the expressed GM1 structures is higher than expected in view of the low proportions evidenced by densitometric quantification. Simultaneous fragmentation of meningioma-associated GM1 (d18:1/24:1) and GM1 (d18:1/24:0) by MS/MS using CID confirmed the postulated structures of the ceramide moieties and provided data on the glycan core, which document that for each of the GM1 (d18:1/24:1) and GM1 (d18:1/24:0) forms both GM1a and GM1b isomers are expressed in the investigated meningioma tissue.     

Nano-electrospray ionization time-of-flight mass spectrometry of gangliosides from human brain tissue

A general approach for the detection and structural elucidation of brain ganglioside species GM1, GD1 and GT1 by nano-electrospray ionization quadrupole time-of-flight (nanoESI-QTOF) mass spectrometry (MS), using combined data from MS and MS/MS analysis of isolated native ganglioside fractions in negative ion mode and their permethylated counterparts in the positive ion mode is presented. This approach was designed to detect and sequence gangliosides present in preparatively isolated ganglioside fractions from pathological brain samples available in only very limited amounts. In these fractions mixtures of homologue and isobaric structures are present, depending on the ceramide composition and the position of the sialic acid attachment site. The interpretation of data for the entire sequence, derived from A, B, C and Y ions by nanoESI-QTOFMS/MS in the negative ion mode of native fractions, can be compromised by ions arising from double and triple internal cleavages. To distinguish between isobaric carbohydrate structures in gangliosides, such as monosialogangliosides GM1a and GM1b, disialogangliosides GD1a, GD1b and GD1c or trisialogangliosides GT1b, GT1c and GT1d, the samples were analysed after permethylation in the positive ion nanoESI-QTOFMS/MS mode, providing set of data, which allows a clear distinction for assignment of outer and inner fragment ions according to their m/z values. The fragmentation patterns from native gangliosides obtained by low-energy collision induced dissociation (CID) by nanoESI-QTOF show common behaviour and follow inherent rules. The combined set of data from the negative and positive ion mode low-energy CID can serve for the detection of structural isomers in mixtures, and to trace new, not previously detected, components.     

Alterations in β-Cell Sphingolipid Profile Associated with ER Stress and iPLA2β: Another Contributor to β-Cell Apoptosis in Type 1 Diabetes

Type 1 diabetes (T1D) development, in part, is due to ER stress-induced β-cell apoptosis. Activation of the Ca²⁺-independent phospholipase A₂ beta (iPLA₂β) leads to the generation of pro-inflammatory eicosanoids, which contribute to β-cell death and T1D. ER stress induces iPLA₂β-mediated generation of pro-apoptotic ceramides via neutral sphingomyelinase (NSMase). To gain a better understanding of the impact of iPLA₂β on sphingolipids (SLs), we characterized their profile in β-cells undergoing ER stress. ESI/MS/MS analyses followed by ANOVA/Student’s t-test were used to assess differences in sphingolipids molecular species in Vector (V) control and iPLA₂β-overexpressing (OE) INS-1 and Akita (AK, spontaneous model of ER stress) and WT-littermate (AK-WT) β-cells. As expected, iPLA₂β induction was greater in the OE and AK cells in comparison with V and WT cells. We report here that ER stress led to elevations in pro-apoptotic and decreases in pro-survival sphingolipids and that the inactivation of iPLA₂β restores the sphingolipid species toward those that promote cell survival. In view of our recent finding that the SL profile in macrophages—the initiators of autoimmune responses leading to T1D—is not significantly altered during T1D development, we posit that the iPLA₂β-mediated shift in the β-cell sphingolipid profile is an important contributor to β-cell death associated with T1D.     

Total synthesis of ganglioside GQ1b and the related polysialogangliosides

The first total synthesis of ganglio-series gangliosides GQ1b, GT1b and GD1b, which contain α-sialyl-(2→8)-α-sialic acid residue in the structure, will be described. Glycosylation of 2-(trimethylsilyl)ethyl O-(2-acetamido-6-O-benzyl-2-deoxy-3,4-O-iso-propylidene-β- -galactopyranosyl)-(1→4)-O-(2,6-di-O-benzyl-β- -galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-D-glucopyranoside (7) with methyl [phenyl 5-acetamido-8-O-(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy- -glycero-α- -galacto-2-nonulopyranosylono-1′,9-lactone)-4,7-di-O-acetyl-3,5-dideoxy-2-thio- -glycero- -galacto-2-nonulopyranosid]onate (8) using N-iodosuccinimide (NIS)-trifluoromethanesulfonic acid (TfOH) in acetonitrile gave the protected GD2 pentasaccharide 9, which was converted into the pentasaccharide acceptor 10 by de-O-isopropylidenation. Glycosylation of 10 with methyl thioglycoside derivatives 18, 26, 34 by use of dimethyl(methylthio)sulfonium triflate (DMTST) gave the protected ganglioside oligosaccharides 19, 27 and 35, respectively. Compounds 9, 19, 27 and 35 were transformed into the corresponding α-trichloroacetimidates 13, 22, 30 and 38, via reductive removal of benzyl groups, O-acetylation, selective removal of 2-(trimethylsilyl)ethyl group, and treatment of trichloroacetonitrile. Condensation of the imidates 13, 22, 30 and 38 with (2S,3R,4E)-2-azido-3-O-benzoyl-4-octadecene-1,3-diol (14) gave the corresponding β-glycosides 15, 23, 31 and 39, which were converted, via selective reduction of azido group, coupling with octadecanoic acid, de-O-acylation, and saponification of methyl esters and lactone groups, into the corresponding gangliosides GD2 (17), GD1b (25), GT1b (33) and GQ1b (41).     

Subcritical Extracts from Major Species of Oil-Bearing Roses—A Comparative Chemical Profiling

A comprehensive chemical profiling of 1,1,1,2-tetrafluoroethane (freon R134a) subcritical extracts from the main genotypes of oil-bearing roses, was performed by gas chromatography–mass spectrometry (GC/MS) and gas chromatography with flame ionization detection (GC-FID) in order to reveal the differences in their chemical composition. One hundred and three individual compounds were identified using GC/MS and their quantitative content was determined using GC-FID, representing 89.8, 92.5, 89.7 and 93.7% of the total content of Rosa gallica L., Rosa damascena Mill., Rosa alba L. and Rosa centifolia L. extracts, respectively. The compounds found in the extracts are representatives of the following main chemical classes: mono-, sesqui- and triterpenoids, phenylethanoids and phenylpropanoids and aliphatic hydrocarbons. Fatty acids, esters and waxes were found, as well. The study revealed that 2-phenylethanol is the most abundant component, ranging 9.0–60.9% followed by nonadecane and nonadecene with 5.1–18.0% geraniol (2.9–14.4%), heneicosane (3.1–11.8%), tricosane (0.1–8.6%), nerol (1.3–6.1%) and citronellol (1.7–5.3%). The extracts demonstrate a specific chemical profile, depending on the botanical species—phenylethanoids and phenyl propanoids are the main group for R. damascena, aliphatic hydrocarbons for R. alba and R. centifolia, while both are found in almost equal amounts in R. gallica. The terpenoid compounds show relatively broad variations: monoterpenes—11.9–25.5% with maximum in R. centifolia; sesquiterpenes—0.6–7.0% with maximum in R. gallica and triterpenes—0.4–3.7% with maximum in R. gallica extract.     

Phenolic Constituents,Antioxidant and Cytoprotective Activities,Enzyme Inhibition Abilities of Five Fractions from Vaccinium dunalianum Wight

The bud of Vaccinium dunalianum Wight has been traditionally consumed as health herbal tea by “Yi” people in Yunnan Province, China, which was locally named “Que Zui tea”. This paper studied the chemical constituents of five fractions from Vaccinium dunalianum, and their enzyme inhibitory effects of α-glucosidase and pancreatic lipase, antioxidant activity, and cytoprotective effects on H₂O₂-induced oxidative damage in HepG2 cells. The methanol extract of V. dunalianum was successively partitioned with petroleum ether (PF), chloroform (CF), ethyl acetate (EF), n-butanol (BF), and aqueous (WF) to obtain five fractions. The chemical profiling of the five fractions was analyzed by ultra-high-performance liquid chromatography coupled with a tandem mass spectrometry (UHPLC-MS/MS), and 18 compounds were tentatively identified. Compared to PF, CF, BF and WF, the EF revealed the highest total phenols (TPC) and total flavonoids (TFC), and displayed the strongest enzyme inhibition ability (α-glucosidase and pancreatic lipase) and antioxidant capacity (DPPH, ABTS and FRAP). Furthermore, these five fractions, especially EF, could effectively inhibit reactive oxygen species (ROS) production and cell apoptosis on H₂O₂-induced oxidative damage protection in HepG2 cells. This inhibitory effect might be caused by the up-regulation of intracellular antioxidant enzyme activity (CAT, SOD, and GSH). The flavonoids and phenolic acids of V. dunalianum might be the bioactive substances responsible for enzyme inhibitory, antioxidant, and cytoprotective activities.     

Novel Fast Chromatography-Tandem Mass Spectrometric Quantitative Approach for the Determination of Plant-Extracted Phytosterols and Tocopherols

Phytosterols and tocopherols are commonly used in food and pharmaceutical industries for their health benefits. Current analysis methods rely on conventional liquid chromatography, using an analytical column, which can be tedious and time consuming. However, simple, and fast analytical methods can facilitate their qualitative and quantitative analysis. In this study, a fast chromatography-tandem mass spectrometric (FC-MS/MS) method was developed and validated for the quantitative analysis of phytosterols and tocopherols. Omitting chromatography by employing flow injection analysis—mass spectrometry (FIA-MS) failed in the quantification of target analytes due to analyte-to-analyte interferences from phytosterols. These interferences arise from their ambiguous MS fingerprints that would lead to false identification and inaccurate quantification. Therefore, a C18 guard column with a 1.9 µm particle size was employed for FC-MS/MS under isocratic elution using acetonitrile/methanol (99:1 v/v) at a flow rate of 600 µL/min. Analyte-to-analyte interferences were identified and eliminated. The false peaks could then be easily identified due to chromatographic separation. In addition, two internal standards were evaluated, namely cholestanol and deuterated cholesterol. Both internal standards contributed to the observed analyte-to-analyte interferences; however, adequate shift in the retention time for deuterated cholesterol eliminated its interferences and allowed for an accurate quantification. The method is fast (1.3 min) compared to published methods and can distinguish false peaks observed in FIA-MS. Seven analytes were quantified simultaneously, namely brassicasterol, campesterol, stigmasterol, β-sitosterol, α-tocopherol, δ-tocopherol, and γ-tocopherol. The method was successfully applied in the quantitative analysis of phytosterols and tocopherols present in the unsaponifiable matter of canola oil deodorizer distillate (CODD). β-sitosterol and γ-tocopherol were the most abundant phytosterols and tocopherols, respectively.     

Polyphenolic Compounds Extracted and Purified from Buddleja Globosa Hope (Buddlejaceae) Leaves Using Natural Deep Eutectic Solvents and Centrifugal Partition Chromatography

Chemical profiling of Buddleja globosa was performed by high-performance liquid chromatography coupled to electrospray ionization (HPLC-DAD-ESI-IT/MS) and quadrupole time-of-flight high-resolution mass spectrometry (HPLC-ESI-QTOF/MS). The identification of 17 main phenolic compounds in B. globosa leaf extracts was achieved. Along with caffeoyl glucoside isomers, caffeoylshikimic acid and several verbascoside derivatives (β-hydroxyverbascoside and β-hydroxyisoverbascoside) were identified. Among flavonoid compounds, the presence of 6-hydroxyluteolin-7-O-glucoside, quercetin-3-O-glucoside, luteolin 7-O-glucoside, apigenin 7-O-glucoside was confirmed. Campneoside I, forsythoside B, lipedoside A and forsythoside A were identified along with verbascoside, isoverbascoside, eukovoside and martynoside. The isolation of two bioactive phenolic compounds verbascoside and forsythoside B from Buddleja globosa (Buddlejaceae) was successfully achieved by centrifugal partition chromatography (CPC). Both compounds were obtained in one-step using optimized CPC methodology with the two-phase solvent system comprising ethyl acetate-n-butanol-ethanol-water (0.25:0.75:0.1:1, v/v). Additionally, eight Natural Deep Eutectic Solvents (NADESs) were tested for the extraction of polyphenols and compared with 80% methanol. The contents of verbascoside and luteolin 7-O-glucoside after extraction with 80% methanol were 26.165 and 3.206 mg/g, respectively. Among the NADESs tested in this study, proline- citric acid (1:1) and choline chloride-1, 2- propanediol (1:2) were the most promising solvents. With these NADES, extraction yields for verbascoside and luteolin 7-O-glucoside were 51.045 and 4.387 mg/g, respectively. Taken together, the results of this study confirm that CPC enabled the fast isolation of bioactive polyphenols from B. globosa. NADESs displayed higher extraction efficiency of phenolic and therefore could be used as an ecofriendly alternative to classic organic solvents.     

Detecting Protein–Glycolipid Interactions Using Glycomicelles and CaR-ESI-MS

This study reports on the use of the catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay, combined with glycomicelles, as a method for detecting specific interactions between water-soluble proteins and glycolipids (GLs) in aqueous solution. The B subunit homopentamers of cholera toxin (CTB₅) and Shiga toxin type 1 B (Stx1B₅) and the gangliosides GM1, GM2, GM3, GD1a, GD1b, GT1b, and GD2 served as model systems for this study. The CTB₅ exhibits broad specificity for gangliosides and binds to GM1, GM2, GM3, GD1a, GD1b, and GT1b; Stx1B₅ does not recognize gangliosides. The CaR-ESI-MS assay was used to analyze solutions of CTB₅ or Stx1B₅ and individual gangliosides (GM1, GM2, GM3, GD1a, GD1b, GT1b, and GD2) or mixtures thereof. The high affinity interaction of CTB₅ with GM1 was successfully detected. However, the apparent affinity, as determined from the mass spectra, is significantly lower than that of the corresponding pentasaccharide or when GM1 is presented in model membranes such as nanodiscs. Interactions between CTB₅ and the low affinity gangliosides GD1a, GD1b, and GT1b, as well as GD2, which served as a negative control, were detected; no binding of CTB₅ to GM2 or GM3 was observed. The CaR-ESI-MS results obtained for Stx1B₅ reveal that nonspecific protein-ganglioside binding can occur during the ESI process, although the extent of binding varies between gangliosides. Consequently, interactions detected for CTB₅ with GD1a, GD1b, and GT1b are likely nonspecific in origin. Taken together, these results reveal that the CaR-ESI-MS/glycomicelle approach for detecting protein–GL interactions is prone to false positives and false negatives and must be used with caution.

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Acridine Based N-Acylhydrazone Derivatives as Potential Anticancer Agents: Synthesis,Characterization and ctDNA/HSA Spectroscopic Binding Properties

A series of novel acridine N-acylhydrazone derivatives have been synthesized as potential topoisomerase I/II inhibitors, and their binding (calf thymus DNA—ctDNA and human serum albumin—HSA) and biological activities as potential anticancer agents on proliferation of A549 and CCD-18Co have been evaluated. The acridine-DNA complex 3b (-F) displayed the highest K_b value (K_b = 3.18 × 10³ M⁻¹). The HSA-derivatives interactions were studied by fluorescence quenching spectra. This method was used for the calculation of characteristic binding parameters. In the presence of warfarin, the binding constant values were found to decrease (K_SV = 2.26 M⁻¹, K_b = 2.54 M⁻¹), suggesting that derivative 3a could bind to HSA at Sudlow site I. The effect of tested derivatives on metabolic activity of A549 cells evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or MTT assay decreased as follows 3b(-F) > 3a(-H) > 3c(-Cl) > 3d(-Br). The derivatives 3c and 3d in vitro act as potential dual inhibitors of hTopo I and II with a partial effect on the metabolic activity of cancer cells A594. The acridine-benzohydrazides 3a and 3c reduced the clonogenic ability of A549 cells by 72% or 74%, respectively. The general results of the study suggest that the novel compounds show potential for future development as anticancer agents.