Application of combined high-performance thin-layer chromatography immunostaining and nanoelectrospray ionization quadrupole time-of-flight tandem mass spectrometry to the structural characterization of high- and low-affinity binding ligands of Shiga toxin 1

Shiga toxin 1 (Stx1) represents an AB5 toxin produced by enterohemorrhagic Escherichia coli, which cause gastrointestinal diseases in humans that are often followed by potentially fatal systemic complications, such as acute encephalopathy and hemolytic uremic syndrome. The expression of the preferential Stx1 receptor, Gb3Cer/CD77 (Gal alpha1-4Gal beta1-4Glc beta1-1Cer), is one of the primary determinants of susceptibility to tissue injury. Due to the clinical importance of this life-threatening toxin, a combined strategy of preparative high-performance thin-layer chromatography (HPTLC) overlay assay and mass spectrometry was developed for the detection and structural characterization of Stx1-binding glycosphingolipids (GSLs). A preparation of neutral GSLs from human erythrocytes, comprising 21.4% and 59.1% of the high- and low-affinity Stx1-binding ligands Gb3Cer/CD77 and Gb4Cer, respectively, was separated on silica gel precoated HPTLC plates and probed for the presence of Stx1 receptors. Stx1 positive on the one hand and anti-Gb3Cer/CD77 and anti-Gb4Cer antibody positive bands from parallel reference runs on the other hand were extracted with chloroform/methanol/water (30/60/8, v/v/v). These crude extracts were used without any further purification for a detailed structural analysis by nanoelectrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-QTOF-MS) in the negative ion mode. In all extracts investigated, neutral GSLs were detected as singly charged deprotonated molecular ions, [M-H]-, and neither buffer-derived salt adducts nor coextracted contaminants from the overlay assay procedure or the silica gel layer were observed. For the structural characterization of Stx1- and antibody-binding GSLs low-energy collision-induced dissociation (CID) was applied to high and low abundant receptor species of the crude extracts. All MS/MS spectra obtained contained full series of Y-type ions, B-type ions and additional ions generated by ring cleavages of the sugar moiety. Only analytical quantities in the microgram scale of a single GSL species within the complex GSL mixture were required for the structural MS characterization of Stx1 ligands as Gb3Cer/CD77 and Gb4Cer. This effective combined HPTLC/MS procedure offers a broad range of applications, not only for toxins of bacterial origin, but also for any GSL-binding agents such as plant-derived lectins or human proteins with yet unknown binding specificities.     

High-throughput screening assay for the quantification of Cer d18:1/16:0, d18:1/24:0, d18:1/24:1, d18:1/18:0, d18:1/14:0, d18:1/20:0, and d18:1/22:0 in HepG2 cells using RapidFire mass spectrometry

Ceramides are known to be involved in various biological processes with their physiological levels elevated in various disease conditions such as diabetes, Alzheimer's, atherosclerosis. To facilitate the rapid screening of Cer d18:1/16:0, d18:1/24:0, d18:1/24:1, d18:1/18:0, d18:1/14:0, d18:1/20:0, and d18:1/22:0 inhibition in HepG2 cells, a RapidFire coupled to tandem mass spectrometry (RF–MS/MS) method has been developed. The RF platform provides an automated solid-phase extraction system that gave a throughput of 12.6 s per sample to an MS/MS system using electrospray ionization under the positive ion mode. Chromatographic separation of Cer d18:1/16:0, d18:1/24:0, d18:1/24:1, d18:1/18:0, d18:1/14:0, d18:1/20:0, and d18:1/22:0 was achieved using a ternary gradient on C₈ type E cartridge. The MS/MS ion transitions monitored were 538.2 → 264.2, 650.7 → 264.2, 648.6 → 264.2, 566.4 → 264.2, 510.4 → 264.2, 594.4 → 264.2, 622.5 → 264.2, and 552.3 → 250.2 for Cer d18:1/16:0, d18:1/24:0, d18:1/24:1, d18:1/18:0, d18:1/14:0, d18:1/20:0, d18:1/22:0, and the internal standard (Cer d17:1/18:0), respectively. The RF–MS/MS methodology showed an excellent performance with an average Z′ value of 0.5–0.7. This is the first report of an RF–MS/MS assay for screening of ceramides which is amenable for high-throughput screening.     

A Novel Method for the Production of Glycosphingolipids

Neolacto‐series ganglioside sialylparagloboside (SPG) is a ganglioside species present in various human tissues, and used in many important studies. In this study, four ganglioside analogs, GM3, GD3, SPG, and NeuAc‐Gal‐GlcNAc‐Gal‐GlcNAc‐Gal‐Glc‐Cer, were synthesized by the saccharide‐primer method using MDCK cells and β‐lactoside primer with different aglycons. As compared to former methods for producing SPG, the primer method was rapid and convenient. Moreover, the yield of SPG was much higher than that obtained by former methods. The production of gangliosides with an azido group in the aglycon moiety was also achieved by using MDCK cells.     

New Insights on Non-Enzymatic Oxidation of Ganglioside GM1 Using Mass Spectrometry

Gangliosides are acidic glycosphingolipids that are present in cell membranes and lipid raft domains, being particularly abundant in central nervous systems. They participate in modulating cell membrane properties, cell–cell recognition, cell regulation, and signaling. Disturbance in ganglioside metabolism has been correlated with the development of diseases, such as neurodegenerative diseases, and in inflammation. Both conditions are associated with an increased production of reactive oxidation species (ROS) that can induce changes in the structure of biomolecules, including lipids, leading to the loss or modification of their function. Oxidized phospholipids are usually involved in chronic diseases and inflammation. However, knowledge regarding oxidation of gangliosides is scarce. In order to evaluate the effect of ROS in gangliosides, an in vitro biomimetic model system was used to study the susceptibility of GM1 (Neu5Acα2-3(Galβ1-3GalNAcβ1-4)Galβ1-4Glcβ1Cer) to undergo oxidative modifications. Oxidation of GM1 under Fenton reaction conditions was monitored using high resolution electrospray ionization-mass spectrometry (ESI-MS) and tandem mass spectrometry (ESI-MS/MS). Upon oxidation, GM1 underwent oxidative cleavages in the carbohydrate chain, leading to the formation of other gangliosides GM2 (GalNAcβ1-4Gal(Neu5Acα2-3)1-4Glcβ1Cer), GM3 (Neu5Acα2-3Galβ1-4Glcβ1Cer), asialo-GM1 (Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1Cer), asialo-GM2 (GalNAcβ1-4Galβ1-4Glcβ1Cer), of the small glycolipids lactosylceramide (LacCer), glucosylceramide (GlcCer), and of ceramide (Cer). In addition, oxygenated GM1 and GM2 (as keto and hydroxy derivatives), glycans, oxidized glycans, and oxidized ceramides were also identified. Nonenzymatic oxidation of GM1 under oxidative stress contributes to the generation of other gangliosides that may participate in the imbalance of gangliosides metabolism in vivo, through uncontrolled enzymatic pathways and, consequently, play some role in neurodegenerative processes.

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Profiling changes triggered during maturation of dendritic cells: a lipidomic approach

Lipids are important in several biological processes because they act as signalling and regulating molecules, or, locally, as membrane components that modulate protein function. This paper reports the pattern of lipid composition of dendritic cells (DCs), a cell type of critical importance in inflammatory and immune responses. After activation by antigens, DCs undergo drastic phenotypical and functional transformations, in a process known as maturation. To better characterize this process, changes of lipid profile were evaluated by use of a lipidomic approach. As an experimental model of DCs, we used a foetal skin-derived dendritic cell line (FSDC) induced to mature by treatment with lipopolysaccharide (LPS). The results showed that LPS treatment increased ceramide (Cer) and phosphatidylcholine (PC) levels and reduced sphingomyelin (SM) and phosphatidylinositol (PI) content. Mass spectrometric analysis of a total lipid extract and of each class of lipids revealed that maturation promoted clear changes in ceramide profile. Quantitative analysis enabled identification of an increase in the total ceramide content and enhanced Cer at m/z 646.6, identified as Cer(d18:1/24:1), and at m/z 648.6, identified as Cer(d18:1/24:0). The pattern of change of these lipids give an extremely rich source of data for evaluating modulation of specific lipid species triggered during DC maturation.     

UCHP Method for Oligosaccharide Combinatorial Library Synthesis

A new methodology for oligosaccharide combinatorial library synthesis using a special hydroxy protecting group, the uni-chemo hydroxy protection (UCHP) group, was developed. The UCHP group was composed of oligomeric amino acid derivatives. The amino terminals of UCHP groups were protected by either Boc or Fmoc groups. By using these two types of UCHP, five kinds of trigalactoses [Galβ1-3Galβ1-3Gal, Galβ1-3(Galβ1-4)Gal, Galβ1-4Galβ1-3Gal, Galβ1-3Galβ1-4Gal, and Galβ1-4Galβ1-4Gal] were successfully synthesized on a solid support as a model of oligosaccharide combinatorial library. Each step of all reactions was also successfully monitored using a combination of two colorimetric tests, chloranil and methyl red-DIC.     

Synthesis and biological evaluations of mono‐ and bis‐ferrocene uracil derivatives

Mono‐ ( 3a – 3e and 4a – 4e ) and bis‐ferrocene ( 5a – 5e and 6a – 6e ) conjugated 5‐substituted uracil derivatives that are bridged by 1,2,3‐triazole linker were synthesized. The impact of ferrocene unit and spacer between ferrocene and triazole on radical scavenging potency was observed. Bis‐ferrocenyl uracil derivatives exhibited better antiproliferative activities than their mono‐ferrocenyl analogs. Bis‐ferrocenyl methyl‐ ( 5b ) and halogen‐substituted ( 5e , 6c , and 6d ) uracil derivatives showed pronounced and selective cytostatic activities on colon adenocarcinoma (CaCo‐2) and Burkitt lymphoma (Raji) cells, with higher potency and selectivity than the reference drug 5‐fluorouracil. Generation of reactive oxygen species (ROS) in CaCo‐2 and Raji cells when treated with compounds 5b , 5e , and 6d was observed. Bis‐ferrocenyl 5‐chlorouracil 6c induced significant disruption in mitochondrial membrane potential that is accompanied by activation of apoptosis in CaCo‐2, Raji, and acute lymphoblastic leukemia (CCRF‐CEM) cells, while 6d caused mitochondrial dysfunction and apoptosis induction in CaCo‐2 and Raji cells. Potent antiproliferative activity of 6c and 6d could be associated with mitochondrial membrane potential disruption accompanied by apoptosis induction. Our findings highlighted 6c and 6d with potent and selective antiproliferative activity on CaCo‐2, Raji, and CCRF‐CEM cells that may be associated with targeting cancer cell mitochondria, as a molecular target.     

Differentiation of type 1 and type 2 chain linkages of native glycosphingolipids by positive-ion fast-atom bombardment mass spectrometry with collision-induced dissociation and linked scanning.

Two underivatized glycosphingolipids, Le(b) and Le(y), isomeric in carbohydrate structure (Fuc alpha 1-->2Gal beta 1--> 3[Fuc alpha 1-->4]GlcNAc beta 1-->3Gal beta 1-->4Glc beta 1-->1Cer and Fuc alpha 1-->2Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc beta 1-->3Gal beta 1--> 4Glc beta 1-->1Cer, respectively), were analyzed by positive-ion fast-atom bombardment (FAB) mass spectrometry with high energy collision-induced dissociation (CID) and linked scanning. The two isomers were distinguishable by the abundance of product ions derived from the non-reducing terminal tetrasaccharide fragment via sequential beta-eliminations of vicinally linked saccharide residues. Following earlier studies from other laboratories, which have dealt primarily with positive-ion FAB-CID mass spectrometry of simple model oligosaccharides, these results exemplify the practical application of two-sector methodology to underivatized complex glycoconjugates commonly encountered in the biomedical field.     

Separation and characterisation of sphingoceramides by high-performance liquid chromatography-electrospray ionisation mass spectrometry

We developed a simple and reliable analytical method for the quantification and the characterization of ceramides extracted from biological samples by high-performance liquid chromatography (HPLC) coupled to electrospray ionisation tandem mass spectrometry (ESI/MS/MS). The chromatographic separation of analytes was carried out in a RP8 column, eluting with a methanol-water mixture in gradient elution mode. The separated lipids were detected by total ion monitoring and characterised by MS/MS spectra; quantitative analysis was performed by integrating the extracted ion peaks obtained in the negative ion mode. Good repeatability was obtained for retention time (0.3-2%), peak area ratio (A(S)/A(IS), 2-8%), as well as limit of detection (LOD, 5-26 pg) and quantification (LOQ, 13-53 pg). The method was validated for the analysis of N-palmitoyl-D-erythro-sphingosine (Cer16), N-stearoyl-D-erythro-sphingosine (Cer18), N-tetracosanoyl-D-erythro-sphingosine (N24:0, lignoceric ceramide, Cer24:0), and N-tetracos-15'-enoyl-D-erythro-sphingosine (N24:1, nervonic ceramide, Cer24:1), giving good results. Lipid mixtures, extracted from skin and epidermal cells, were analysed for their content of the studied ceramides.     

Development and validation of LC-MS/MS method for determination of very long acyl chain (C22:0 and C24:0) ceramides in human plasma

Ceramide is a key metabolite in both anabolic and catabolic pathways of sphingolipids. The very long fatty acyl chain ceramides N-(docosanoyl)-sphing-4-enine (Cer(22:0)) and N-(tetracosanoyl)-sphing-4-enine (Cer(24:0)) are associated with multiple biological functions. Elevated levels of these sphingolipids in tissues and in the circulation have been associated with insulin resistance and diabetes. To facilitate quantification of these very long chain ceramides in clinical samples from human subjects, we have developed a sensitive, accurate, and high-throughput assay for determination of Cer(22:0) and Cer(24:0) in human plasma. Cer(22:0) and Cer(24:0) and their deuterated internal standards were extracted by protein precipitation and chromatographically separated by HPLC. The analytes and their internal standards were ionized using positive-ion electrospray mass spectrometry, then detected by multiple-reaction monitoring with a tandem mass spectrometer. Total liquid chromatography–tandem mass spectrometry (LC-MS/MS) runtime was 5 min. The assay exhibited a linear dynamic range of 0.02–4 and 0.08–16 μg/ml for Cer(22:0) and Cer(24:0), respectively, in human plasma with corresponding absolute recoveries from plasma at 109 and 114 %, respectively. The lower limit of quantifications were 0.02 and 0.08 μg/ml for Cer(22:0) and Cer(24:0), respectively. Acceptable precision and accuracy were obtained for concentrations over the calibration curve ranges. With the semi-automated format and short LC runtime for the assay, a throughput of ～200 samples/day can easily be achieved.

Profiling of esterified fatty acids as biomarkers in the blood of dengue fever patients using a microliter‐scale extraction followed by gas chromatography and mass spectrometry

An improved gas chromatography with mass spectrometry procedure was developed to highlight the esterified fatty acids in 100 μL blood of dengue fever patients in the early febrile phase versus healthy volunteers. 24 adult patients and 24 healthy volunteers were included in this study. The recoveries of targeted esterified fatty acids content were in the range of 92.10–101.00% using methanol/dichloromethane (2:1, v/v) as the extraction solvent. An efficient chromatographic separation of targeted 17 esterified fatty acid methyl esters was obtained. The limits of detection and quantification were within the range of 16–131 and 53–430 ng/mL, respectively. The relative standard deviation of intraday and interday precision values ranged from 0.4 to 5.0%. The statistical data treatment showed a significant decrease of the content of four saturated fatty acids, C14:0, C15:0, C16:0, and C18:0 (P value < 0.05), and also showed a decrease of the content of eight unsaturated fatty acids, C16:1, C18:3n6, C18:2n6, C18:1n9, C20:3n3, C20:4n6, C20:2, and C22:6n3 (P value < 0.05) in dengue fever patients. Moreover, the amount of three omega‐6 fatty acids including C18:3n6, C18:2n6, and C20:4n6 was dramatically decreased in the blood of dengue fever patients to a limit of 50 ± 10%.     

Identification of the individual molecular species of ceramides derived from human erythrocytes using HPLC/MS and HPLC/MS/MS

A preparative separation of total ceramide fraction from the crude extract of the erythrocytary lipids was done by means of normal-phase column chromatography. This was followed by comprehensive profiling of the molecular species in the obtained ceramide by means of HPLC/MS and HPLC/MS/MS. The MS/MS analysis displayed that human erythrocytes contain 19 molecular species of the ceramide of which 12 can be unambiguously identified; erythrocytary ceramides may contain not only sphingosine but also sphinganine as their building blocks; one of the species (namely Cer 24:2/S18) previously has managed to escape identification. We also obtained a quantitative profile of major ceramide species showing the prevalence of Cer 22:0/S18, 24:0/S18 and 24:1/S18.     

Simple and convenient synthesis of a fluorinated GM4 analogue

A series of fluorinated galactosides, dodecyl 2-deoxy-2-fluoro-β-d-galactopyranoside (2F Gal), dodecyl 4-deoxy-4-fluoro-β-d-galactopyranoside (4F Gal) and dodecyl 6-deoxy-6-fluoro-β-d-galactopyranoside (6F Gal), was chemically synthesized and introduced to B16 cells to serve as scaffolds for cellular enzyme glycosylation. Results showed that the presence of fluorine exercised significant effects on cell viability. Among the fluorinated galactosides used, 2F Gal was glycosylated to afford a GM4 analogue.     

HPTLC‐MALDI MS for (glyco)sphingolipid multiplexing in tissues and blood: A promising strategy for biomarker discovery and clinical applications

Sphingolipids have hydrophilic and hydrophobic properties, different saturation and combination of the oligosaccharide chains and mass homology of species located in a narrow m/z region hampering their recognition. To target sphingolipids for diagnostic purposes, standardized methods for lipid extraction, quali‐ and quantitative assessments are required. In this study, HPTLC‐MALDI MS was adopted to establish sphingolipid and glycosphingolipid profiles in muscle, brain and serum to create a database of molecules to be searched in the preclinical and clinical investigations. Specific protocols for lipid extraction were set up based on the characteristics of the tissue or/and fluids; this approach maximizes the HPTLC‐MALDI MS analytical throughput both for lipids extracted in organic and aqueous phase. This study indicates that alkaline hydrolysis is necessary for the detection of low abundant species such as Gb3Cer and ceramides in serum and Gb4Cer, CerP and HexCer in muscle tissue. The high hydrophobicity of ceramides has been overcome by the development of HPTLC plate in chloroform:methanol/50:3.5, which increases the number and the intensity of low abundant Cer species. MS/MS analysis has been conducted directly on HPTLC plate allowing the molecular recognition; furthermore a dataset of spectra was acquired to create a database for future profiling of these molecules.     

Synthesis and Characterization of Sulfated Gal‐β‐1,3/4‐GlcNAc Disaccharides through Consecutive Protection/Glycosylation Steps

We have developed an expeditious procedure to yield large amounts of orthogonally protected Gal‐β1,3/4‐GlcNAc, which allowed for the systematic introduction of a sulfate group onto the C3/C6 positions of Gal and/or the C6 position of GlcNAc. In particular, the disaccharide precursors were prepared in five or six steps and high overall yield from para‐tolyl‐6‐O‐tert‐butyldiphenylsilyl‐1‐thio‐β‐D ‐galactopyranoside. After deprotection and sulfation steps, the final products were characterized by using several NMR methods to unambiguously confirm the location of each introduced sulfate group and they were examined for their binding specificity of human galectin‐1 and galectin‐8.     

Novel Antileukemic Sterol Glycosides from Ajuga salicifolia

Two novel and three new sterol glycosides were isolated from the MeOH extract of the aerial parts of Ajuga salicifolia (L.) Schreber . The structures of the compounds were elucidated as (3R,16S,17S,20R,22S,23S, 24S,25S)‐16,23 : 16,27 : 22,25‐triepoxy‐3‐(β‐D ‐glucopyranosyloxy)coprostigmast‐7‐en‐17‐ol ( 1 ), (3R,16S,17S, 20R,22S,23S,24S,25S)‐16,23 : 16,27 : 22,25‐triepoxy‐3‐{[β‐D ‐glucopyranosyl‐(1→2)‐β‐D ‐glucopyranosyl]oxy}coprostigmast‐7‐en‐17‐ol ( 2 ), (3R,16S,17R,20S,22R,24S,25S)‐22,25‐epoxy‐3,27‐bis(β‐D ‐glucopyranosyloxy)coprostigmast‐7‐en‐16‐ol ( 3 ), (3R,16S,17R,20S,22R,24S,25S)‐22,25‐epoxy‐3‐{[β‐D ‐glucopyranosyl‐(1→2)‐β‐D ‐glucopyranosyl]oxy}‐27‐(β‐D ‐glucopyranosyloxy)coprostigmast‐7‐en‐16‐ol ( 4 ), and (3R,16R,17S,20R,22S,23S, 24S,25S)‐22,25‐epoxy‐3‐(β‐D ‐glucopyranosyloxy)coprostigmast‐7‐ene‐16,17,23,27‐tetrol 27‐acetate ( 5 ) by means of 1D and 2D NMR spectroscopy and HR‐MALDI mass spectrometry. The novel compounds, which consist of three additional ring systems at the coprostigmastane skeleton, were named ajugasalicioside A ( 1 ) and B ( 2 ), and the new compounds C ( 3 ), D ( 4 ) and E ( 5 ). In our cytotoxicity assays (HeLa cells, Jurkat T cells, and peripheral mononuclear blood cells), ajugasaliciosides A–D specifically inhibited the viability and growth of Jurkat T‐leukemia cells at concentrations below 10 μM . Ajugasalicioside A ( 1 ; (IC₅₀=6 μM ) and C ( 3 ; IC₅₀=3 μM ) were the most active compounds. Ajugasalicioside A ( 1 ) induced cell‐cell contact, inhibited Jurkat T cell proliferation, and up‐regulated mRNA levels of the cell‐cycle regulator cyclin D1, which might be an indication for cell differentiation. Furthermore, 1 down‐regulated the mRNA levels of the NF‐κB subunit p65 in a concentration‐dependent manner. These effects were not found for ajugasalicioside B ( 2 ), which has an additional glucose unit, and the onset of cytotoxicity of 2 (IC₅₀=10 μM ) was delayed by 24 h.     

Capillary electrochromatography with novel stationary phases. IV. Retention behavior of glycosphingolipids on porous and non-porous octadecyl sulfonated silica

In this investigation, capillary electrochromatography (CEC) with a novel stationary phase proved useful for the separation of neutral and acidic glycosphingolipids (GSLs). Four different gangliosides, namely G(M1a), G(D1a), G(D1b) and G(T1b), served as the acidic GSLs model solutes. The following four GSLs: galactosylceramide (GalCer), lactosylceramide (LacCer), globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) served as the typical neutral GSLs. The stationary phase, octadecyl sulfonated silica (ODSS), consisted of octadecyl functions bonded to a negatively charged layer containing sulfonic acid groups. Porous and non-porous ODSS stationary phases were examined. The retention behavior of the acidic and neutral GSLs was examined over a wide range of elution conditions, including the nature of the electrolyte and organic modifier and the pH of the mobile phase. The porous ODSS stationary phase yielded the separation of the four different gangliosides using a hydro-organic eluent of moderate eluent strength whereas the non-porous ODSS stationary phase permitted the separation of the four neutral GSLs with a mobile phase of relatively high eluent strength.     

Further Withaphysalin Derivatives from Acnistus arborescens

Two new epimeric chlorinated withaphysalins, rel‐(4β,5β,6α,18S,22R)‐ and rel‐(4β,5β,6α,18R,22R)‐6‐chloro‐18,20‐epoxy‐18‐ethoxy‐4,5‐dihydroxy‐1‐oxowitha‐2,24‐diene‐26,22‐lactone ( 1 and 2 resp.), together with the new rel‐(4β,5β,6α,18R,22R)‐6‐chloro‐18,20‐epoxy‐4,5‐dihydroxy‐18‐methoxy‐1‐oxowitha‐2,24‐diene‐26,22‐lactone ( 3 ) and rel‐(3β,4β,5β,6β,18R,22R)‐5,6:18,20‐diepoxy‐3,18‐diethoxy‐4‐hydroxy‐1‐oxowith‐24‐ene‐26,22‐lactone ( 4 ) were isolated from the leaves of Acnistus arborescens and named withaphysalins T–W, respectively. The final structures and the complete ¹H‐ and ¹³C‐NMR assignments of the three chlorowithaphysalins 1 – 3 were performed by means of HR‐ESI‐MS and 1D‐ and 2D‐NMR experiments, including COSY, HSQC, and HMBC, beside comparison with spectral data of analogous compounds from the literature. The structure of 4 was also confirmed by means of a single‐crystal X‐ray diffraction analysis.     

Highly Polar Triterpenoid Saponins from the Roots of Saponaria officinalis L.

Five new triterpenoid saponins, including 3‐O‐β‐d ‐galactopyranosyl‐(1→2)‐[β‐d ‐xylopyranosyl‐(1→3)]‐β‐d ‐glucuronopyranosyl quillaic acid 28‐O‐β‐d ‐glucopyranosyl‐(1→3)‐β‐d ‐xylopyranosyl‐(1→4)‐α‐l ‐rhamnopyranosyl‐(1→2)‐[β‐d ‐xylopyranosyl‐(1→3)‐(4‐O‐acetyl)‐β‐d ‐quinovopyranosyl‐(1→4)]‐β‐d ‐fucopyranoside ( 1 ), 3‐O‐β‐d ‐galactopyranosyl‐(1→2)‐[β‐d ‐xylopyranosyl‐(1→3)]‐β‐d ‐glucuronopyranosyl quillaic acid 28‐O‐(6‐O‐acetyl)‐β‐d ‐glucopyranosyl‐(1→3)‐[β‐d ‐xylopyranosyl‐(1→4)]‐α‐l ‐rhamnopyranosyl‐(1→2)‐[β‐d ‐xylopyranosyl‐(1→3)‐(4‐O‐acetyl)‐β‐d ‐quinovopyranosyl‐(1→4)]‐β‐d ‐fucopyranoside ( 2 ), 3‐O‐β‐d ‐galactopyranosyl‐(1→2)‐[β‐d ‐xylopyranosyl‐(1→3)]‐β‐d ‐glucuronopyranosyl quillaic acid 28‐O‐β‐d ‐xylopyranosyl‐(1→4)‐α‐l ‐rhamnopyranosyl‐(1→2)‐[β‐d ‐xylopyranosyl‐(1→3)‐(4‐O‐acetyl)‐β‐d ‐quinovopyranosyl‐(1→4)]‐β‐d ‐fucopyranoside ( 3 ), 3‐O‐β‐d ‐galactopyranosyl‐(1→2)‐[β‐d ‐xylopyranosyl‐(1→3)]‐β‐d ‐glucuronopyranosyl quillaic acid 28‐O‐β‐d ‐glucopyranosyl‐(1→3)‐β‐d ‐xylopyranosyl‐(1→4)‐α‐l ‐rhamnopyranosyl‐(1→2)‐[(4‐O‐acetyl)‐β‐d ‐quinovopyranosyl‐(1→4)]‐β‐d ‐fucopyranoside ( 4 ), 3‐O‐β‐d ‐galactopyranosyl‐(1→2)‐[β‐d ‐xylopyranosyl‐(1→3)]‐β‐d ‐glucuronopyranosyl quillaic acid 28‐O‐(6‐O‐acetyl)‐β‐d ‐glucopyranosyl‐(1→3)‐[β‐d ‐xylopyranosyl‐(1→4)]‐α‐l ‐rhamnopyranosyl‐(1→2)‐[(4‐O‐acetyl)‐β‐d ‐quinovopyranosyl‐(1→4)]‐β‐d ‐fucopyranoside ( 5 ) together with two known congeners, saponariosides A ( 6 ) and B ( 7 ) were isolated from the roots of Saponaria officinalis L. Their structures were elucidated by extensive spectroscopic methods, including 1D‐ (¹H, ¹³C) and 2D‐NMR (DQF‐COSY, TOCSY, HSQC, and HMBC) experiments, HR‐ESI‐MS, and acid hydrolysis.