Phytoceramides from the Marine Sponge Monanchora clathrata: Structural Analysis and Cytoprotective Effects.

In our research on sphingolipids from marine invertebrates, a mixture of phytoceramides was isolated from the sponge Monanchora clathrata (Western Australia). Total ceramide, ceramide molecular species (obtained by RP-HPLC, high-performance liquid chromatography on reversed-phase column) and their sphingoid/fatty acid components were analyzed by NMR (nuclear magnetic resonance) spectroscopy and mass spectrometry. Sixteen new (1b, 3a, 3c, 3d, 3f, 3g, 5c, 5d, 5f, 5g, 6b-g) and twelve known (2b, 2e, 2f, 3b, 3e, 4a-c, 4e, 4f, 5b, 5e) compounds were shown to contain phytosphingosine-type backbones i-t17:0 (1), n-t17:0 (2), i-t18:0 (3), n-t18:0 (4), i-t19:0 (5), or ai-t19:0 (6), N-acylated with saturated (2R)-2-hydroxy C21 (a), C22 (b), C23 (c), i-C23 (d), C24 (e), C25 (f), or C26 (g) acids. The used combination of the instrumental and chemical methods permitted the more detailed investigation of the sponge ceramides than previously reported. It was found that the cytotoxic effect of crambescidin 359 (alkaloid from M. clathrata) and cisplatin decreased after pre-incubation of MDA-MB-231 and HL-60 cells with the investigated phytoceramides. In an in vitro paraquat model of Parkinson's disease, the phytoceramides decreased the neurodegenerative effect and ROS (reactive oxygen species) formation induced by paraquat in neuroblastoma cells. In general, the preliminary treatment (for 24 or 48 h) of the cells with the phytoceramides of M. clathrata was necessary for their cytoprotective functions, otherwise the additive damaging effect of these sphingolipids and cytotoxic compounds (crambescidin 359, cisplatin or paraquat) was observed.

Dietary Ceramide Prepared from Soy Sauce Lees Improves Skin Barrier Function in Hairless Mice.

Dietary sphingolipids such as glucosylceramide and sphingomyelin are known to improve the skin barrier function of damaged skin. In this study, we focused on free-ceramide prepared from soy sauce lees, which is a byproduct of soy sauce production. The effects of dietary soy sauce lees ceramide on the skin of normal mice were evaluated and compared with those of dietary maize glucosylceramide. We found that transepidermal water loss value was significantly suppressed by dietary supplementation with soy sauce lees ceramide as effectively as or more effectively than maize glucosylceramide. Although the content of total and each subclass of ceramide in the epidermis was not significantly altered by dietary sphingolipids, that of 12 types of ceramide molecules, which were not present in dietary sources, was significantly increased upon ingestion of maize glucosylceramide and showed a tendency to increase with soy sauce lees ceramide intake. In addition, the mRNA expression of ceramide synthase 4 and involucrin in the skin was downregulated by sphingolipids. This study, for the first time, demonstrated that dietary soy sauce lees ceramide enhances skin barrier function in normal hairless mice, although further studies are needed to clarify the molecular mechanism.

Determination of the lipid composition of the GPI anchor.

In eukaryotic cells, a subset of cell surface proteins is attached by the glycolipid glycosylphosphatidylinositol (GPI) to the external leaflet of the plasma membrane where they play important roles as enzymes, receptors, or adhesion molecules. Here we present a protocol for purification and mass spectrometry analysis of the lipid moiety of individual GPI-anchored proteins (GPI-APs) in yeast. The method involves the expression of a specific GPI-AP tagged with GFP, solubilization, immunoprecipitation, separation by electrophoresis, blotting onto PVDF, release and extraction of the GPI-lipid moiety and analysis by mass spectrometry. By using this protocol, we could determine the precise GPI-lipid structure of the GPI-AP Gas1-GFP in a modified yeast strain. This protocol can be used to identify the lipid composition of the GPI anchor of distinct GPI-APs from yeast to mammals and can be adapted to determine other types of protein lipidation.

UHPLC-Q-Orbitrap HRMS-based quantitative lipidomics reveals the chemical changes of phospholipids during thermal processing methods of Tan sheep meat.

Thermal processing affects the lipid compositions of meat products. The study determined the effects of boiled, steamed and roasted processing methods on the lipidomics profiles of Tan sheep meat with a validated UPLC-Q-Orbitrap HRMS combined lipid screening strategy method. Combined with sphingolipid metabolism, the boiled approach was the suitable choice for atherosclerosis patients for more losses of sphingomyelin than ceramide in meat. The similarly less losses of phosphatidylcholine and lysophosphatidylcholine showed in glycerophospholipid metabolism implied that steamed Tan sheep meat was more suitable for the populations of elderly and infants. Furthermore, a total of 90 lipids with significant difference (VIP > 1) in 6 lipid subclasses (sphingomyelin, ceramide, lysophosphatidylcholine, phosphatidylcholine, phosphatidylethanolamines, triacylglycerol,) were quantified among raw and three types of thermal processed Tan sheep meat, further providing useful information for identification of meat products with different thermal processing methods (LOD with 0.14-0.31 µg kg-1, LOQ with 0.39-0.90 µg kg-1).

Protocol for measuring sphingolipid metabolism in budding yeast.

Sphingolipid biosynthesis occurs in both the endoplasmic reticulum (ER) and the Golgi apparatus. Ceramide synthesized in the ER is transported to the Golgi and incorporated into complex sphingolipids. Here, we present a step-by-step protocol to analyze sphingolipid metabolism in budding yeast. Ceramide and inositolphosphorylceramide (IPC) are classes of sphingolipids present in yeast and are metabolically labeled with radioactive precursors. This protocol for metabolic labeling can be used to investigate ceramide transport in an in vivo environment. For complete details on the use and execution of this protocol, please refer to Ikeda et al. (2020).

Molecular docking, SAR analysis and biophysical approaches in the study of the antibacterial activity of ceramides isolated from Cissus incisa.

The developing of antibacterial resistance is becoming in crisis. In this sense, natural products play a fundamental role in the discovery of antibacterial agents with diverse mechanisms of action. Phytochemical investigation of Cissus incisa leaves led to isolation and characterization of the ceramides mixture (1): (8E)-2-(tritriacont-9-enoyl amino)-1,3,4-octadecanetriol-8-ene (1-I); (8E)-2-(2',3'-dihydroxyoctacosanoyl amino)-1,3,4-octadecanetriol-8-ene (1-II); (8E)-2-(2'-hydroxyheptacosanoyl amino)-1,3,4-octadecanetriol-8-ene (1-III); and (8E)-2-(-2'-hydroxynonacosanoyl amino)-1,3,4-octadecanetriol-8-ene (1-IV). Until now, this is the first report of the ceramides (1-I), (1-II), and (1-IV). The structures were elucidated using NMR and mass spectrometry analyses. Antibacterial activity of ceramides (1) and acetylated derivates (2) was evaluated against nine multidrug-resistant bacteria by Microdilution method. (1) showed the best results against Gram-negatives, mainly against carbapenems-resistant Acinetobacter baumannii with MIC = 50 µg/mL. Structure-activity analysis and molecular docking revealed interactions between plant ceramides with membrane proteins, and enzymes associated with biological membranes of Gram-negative bacteria, through hydrogen bonding of functional groups. Vesicular contents release assay showed the capacity of (1) to disturb membrane permeability detected by an increase of fluorescence probe over time. The membrane disruption is not caused for ceramides lytic action on cell membranes, according in vitro hemolyticactivity results. Combining SAR analysis, bioinformatics and biophysical techniques, and also experimental tests, it was possible to explain the antibacterial action of these natural ceramides.

Terminaliamide, a new ceramide and other phytoconstituents from the roots of Terminalia mantaly H. Perrier and their biological activities.

Terminaliamide (1), a new ceramide was isolated from the roots of Terminalia mantaly H. Perrier (Combretaceae) along with 4 known compounds (2-5). The structures of the compounds were elucidated using 1D and 2D NMR spectroscopy analysis and mass spectrometry. Compound 1 exhibited moderated antibacterial activity towards Staphylococcus aureus with MIC value of 62.5 µg/mL. The crude MeOH extract (TMr) highly reduced Plasmodium falciparum growth with an IC50 value of 10.11 µg/mL, while hexane fraction (F1) highly reduced Trypanosoma brucei brucei growth with an IC50 value of 5.60 µg/mL. All tested samples presented little or no in vitro cytotoxicity on HeLa cell line. The present work confirms that T. mantaly is medicinally important and may be used effectively as an antimicrobial, an antiplasmodial and an antitrypanosomial with promising therapeutic index.

Obese Mice with Dyslipidemia Exhibit Meibomian Gland Hypertrophy and Alterations in Meibum Composition and Aqueous Tear Production.

BACKGROUND: Dyslipidemia may be linked to meibomian gland dysfunction (MGD) and altered meibum lipid composition. The purpose was to determine if plasma and meibum cholesteryl esters (CE), triglycerides (TG), ceramides (Cer) and sphingomyelins (SM) change in a mouse model of diet-induced obesity where mice develop dyslipidemia. METHODS: Male C57/BL6 mice (8/group, age = 6 wks) were fed a normal (ND; 15% kcal fat) or an obesogenic high-fat diet (HFD; 42% kcal fat) for 10 wks. Tear production was measured and meibography was performed. Body and epididymal adipose tissue (eAT) weights were determined. Nano-ESI-MS/MS and LC-ESI-MS/MS were used to detect CE, TG, Cer and SM species. Data were analyzed by principal component analysis, Pearson's correlation and unpaired t-tests adjusted for multiple comparisons; significance set at p ≤ 0.05. RESULTS: Compared to ND mice, HFD mice gained more weight and showed heavier eAT and dyslipidemia with higher levels of plasma CE, TG, Cer and SM. HFD mice had hypertrophic meibomian glands, increased levels of lipid species acylated by saturated fatty acids in plasma and meibum and excessive tear production. CONCLUSIONS: The majority of meibum lipid species with saturated fatty acids increased with HFD feeding with evidence of meibomian gland hypertrophy and excessive tearing. The dyslipidemia is associated with altered meibum composition, a key feature of MGD.

Isolation of glycosylinositol phosphoceramide and phytoceramide 1-phosphate in plants and their chemical stabilities.

Glycosylinositol phosphoceramide (GIPC) is a sphingophospholipid in plants. Recently, we identified that GIPC is hydrolyzed to phytoceramide 1-phosphate (PC1P) by an uncharacterized phospholipase D activity following homogenization of certain plant tissues. We now developed methods for isolation of GIPC and PC1P from plant tissues and characterized their chemical stabilities. Hydrophilic solvents, namely a lower layer of a mixed solvent system consisting of isopropanol/hexane/water (55:20:25, v/v/v) was efficient solvent for extraction and eluent in column chromatography. GIPC was isolated by Sephadex column chromatography followed by TLC. A conventional method, such as the Bligh and Dyer method, was applicable for PC1P extraction. Specifically, PC1P was isolated by TLC following mild alkali treatment of lipid extracts of plants. The yields of GIPC and PC1P in our methods were both around 50-70%. We found that PC1P is tolerant against heat (up to 125 °C), strong acid (up to 10 M HCl), and mild alkali (0.1 M KOH). In contrast, significant degradation of GIPC occurred at 100 °C and 1.0 M HCl treatment, suggesting the instability of the inositol glycan moiety in these conditions. These data will be useful for further biochemical and nutritional studies on these sphingolipids.

New Cytotoxic Natural Products from the Red Sea Sponge Stylissa carteri.

Bioactivity-guided isolation supported by LC-HRESIMS metabolic profiling led to the isolation of two new compounds, a ceramide, stylissamide A (1), and a cerebroside, stylissoside A (2), from the methanol extract of the Red Sea sponge Stylissa carteri. Structure elucidation was achieved using spectroscopic techniques, including 1D and 2D NMR and HRMS. The bioactive extract's metabolomic profiling showed the existence of various secondary metabolites, mainly oleanane-type saponins, phenolic diterpenes, and lupane triterpenes. The in vitro cytotoxic activity of the isolated compounds was tested against two human cancer cell lines, MCF-7 and HepG2. Both compounds, 1 and 2, displayed strong cytotoxicity against the MCF-7 cell line, with IC50 values at 21.1 ± 0.17 µM and 27.5 ± 0.18 µM, respectively. They likewise showed a promising activity against HepG2 with IC50 at 36.8 ± 0.16 µM for 1 and IC50 30.5 ± 0.23 µM for 2 compared to the standard drug cisplatin. Molecular docking experiments showed that 1 and 2 displayed high affinity to the SET protein and to inhibitor 2 of protein phosphatase 2A (I2PP2A), which could be a possible mechanism for their cytotoxic activity. This paper spreads light on the role of these metabolites in holding fouling organisms away from the outer surface of the sponge, and the potential use of these defensive molecules in the production of novel anticancer agents.

Establishment of a Measurement System for Sphingolipids in the Cerebrospinal Fluid Based on Liquid Chromatography-Tandem Mass Spectrometry, and Its Application in the Diagnosis of Carcinomatous Meningitis.

OBJECTIVES: Sphingolipids have been demonstrated to be involved in many human diseases. However, measurement of sphingolipids, especially of sphingosine 1-phosphate (S1P) and dihydro-sphingosine 1-phosphate (dhS1P), in blood samples requires strict sampling, since blood cells easily secrete these substances during sampling and storage, making it difficult to introduce measurement of sphingolipids in clinical laboratory medicine. On the other hand, cerebrospinal fluid (CSF) contains few blood cells. Therefore, we attempted to establish a system based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the measurement of sphingolipids in the CSF, and applied it for the diagnosis of carcinomatous meningitis. METHODS: We developed and validated a LC-MS/MS-based measurement system for S1P and dhS1P and for ceramides and sphingosines, used this system to measure the levels of these sphingolipids in the CSF collected from the subjects with cancerous meningitis, and compared the levels with those in normal routine CSF samples. RESULTS: Both the measurement systems for S1P/dhS1P and for ceramides/sphingosines provided precision with the coefficient of variation below 20% for sphingolipids in the CSF samples. We also confirmed that the levels of S1P, as well as ceramides/sphingosines, in the CSF samples did not increase after the sampling. In the CSF samples collected from patients with cancerous meningitis, we observed that the ratio of S1P to ceramides/sphingosine and that of dhS1P to dihydro-sphingosine were higher than those in control samples. CONCLUSIONS: We established and validated a measurement system for sphingolipids in the CSF. The system offers promise for being introduced into clinical laboratory testing.

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 Targeted Mass Spectrometric Analysis Reveals the Presence of a Reduced but Dynamic Sphingolipid Metabolic Pathway in an Ancient Protozoan, Giardia lamblia.

Giardia lamblia, a single-celled eukaryote, colonizes and thrives in the small intestine of humans. Because of its compact and reduced genome, Giardia has adapted a "minimalistic" life style, as it becomes dependent on available resources of the small intestine. Because Giardia expresses fewer sphingolipid (SL) genes-and glycosphingolipids are critical for encystation-we investigated the SL metabolic cycle in this parasite. A tandem mass spectrometry (MS/MS) analysis reveals that major SLs in Giardia include sphingomyelins, sphingoid bases, ceramides, and glycosylceramides. Many of these lipids are obtained by Giardia from the growth medium, remodeled at their fatty acyl chains and end up in the spent medium. For instance, ceramide-1-phosphate, a proinflammatory molecule that is not present in the culture medium, is generated from sphingosine (abundant in the culture medium) possibly by remodeling reactions. It is then subsequently released into the spent medium. Thus, the secretion of ceramide-1-phospate and other SL derivatives by Giardia could be associated with inflammatory bowel disease observed in acute giardiasis. Additionally, we found that the levels of SLs increase in encysting Giardia and are differentially regulated throughout the encystation cycle. We propose that SL metabolism is important for this parasite and, could serve as potential targets for developing novel anti-giardial agents.

A metabolic perspective on CSF-mediated neurodegeneration in multiple sclerosis.

Multiple sclerosis is an autoimmune demyelinating disorder of the CNS, characterized by inflammatory lesions and an underlying neurodegenerative process, which is more prominent in patients with progressive disease course. It has been proposed that mitochondrial dysfunction underlies neuronal damage, the precise mechanism by which this occurs remains uncertain. To investigate potential mechanisms of neurodegeneration, we conducted a functional screening of mitochondria in neurons exposed to the CSF of multiple sclerosis patients with a relapsing remitting (n = 15) or a progressive (secondary, n = 15 or primary, n = 14) disease course. Live-imaging of CSF-treated neurons, using a fluorescent mitochondrial tracer, identified mitochondrial elongation as a unique effect induced by the CSF from progressive patients. These morphological changes were associated with decreased activity of mitochondrial complexes I, III and IV and correlated with axonal damage. The effect of CSF treatment on the morphology of mitochondria was characterized by phosphorylation of serine 637 on the dynamin-related protein DRP1, a post-translational modification responsible for unopposed mitochondrial fusion in response to low glucose conditions. The effect of neuronal treatment with CSF from progressive patients was heat stable, thereby prompting us to conduct an unbiased exploratory lipidomic study that identified specific ceramide species as differentially abundant in the CSF of progressive patients compared to relapsing remitting multiple sclerosis. Treatment of neurons with medium supplemented with ceramides, induced a time-dependent increase of the transcripts levels of specific glucose and lactate transporters, which functionally resulted in progressively increased glucose uptake from the medium. Thus ceramide levels in the CSF of patients with progressive multiple sclerosis not only impaired mitochondrial respiration but also decreased the bioavailability of glucose by increasing its uptake. Importantly the neurotoxic effect of CSF treatment could be rescued by exogenous supplementation with glucose or lactate, presumably to compensate the inefficient fuel utilization. Together these data suggest a condition of 'virtual hypoglycosis' induced by the CSF of progressive patients in cultured neurons and suggest a critical temporal window of intervention for the rescue of the metabolic impairment of neuronal bioenergetics underlying neurodegeneration in multiple sclerosis patients.

Isolation of Ceramides from Tagetes patula L. Yellow Flowers and Nematicidal Activity of the Fractions and Pure Compounds against Cyst Nematode, Heterodera zeae.

Investigation of yellow flower extract of Tagetes patula L. led to the identification of an aggregate of five phytoceramides. Among them, (2R)-2-hydroxy-N-[(2S,3S,4R,8E)-1,3,4-trihydroxyicos-8-en-2-yl]icosanamide, (2R)-2-hydroxy-N-[(2S,3S,4R,8E)-1,3,4-trihydroxyicos-8-en-2-yl]heneicosanamide, (2R)-2-hydroxy-N-[(2S,3S,4R,8E)-1,3,4-trihydroxyicos-8-en-2-yl]docosanamide, and (2R)-2-hydroxy-N-[(2S,3S,4R,8E)-1,3,4-trihydroxyicos-8-en-2-yl]tricosanamide were identified as new compounds and termed as tagetceramides, whereas (2R)-2-hydroxy-N-[(2S,3S,4R,8E)-1,3,4-trihydroxyicos-8-en-2-yl]tetracosanamide was a known ceramide. A steroid (ß-sitosterol glucoside) was also isolated from the subsequent fraction. The structures of these compounds were determined on the basis of spectroscopic analyses, as well as chemical method. Several other compounds were also identified by GC/MS analysis. The fractions and some commercial products, a ceramide HFA, ß-sitosterol, and stigmasterol were evaluated against an economically important cyst nematode, Heterodera zeae. Ceramide HFA showed 100 % mortality, whereas, ß-sitosterol and stigmasterol were 40-50 % active, at 1 % concentration after 24 h of exposure time, while ß-sitosterol glucoside revealed no activity against the nematode.

The effect of high fat diet and metformin treatment on liver lipids accumulation and their impact on insulin action.

We sought to determine whether metformin treatment reverses a high-fat diet (HFD)-induced hepatic insulin resistance (IRes) and to identify lipid intermediates involved in induction of liver IRes. The experiments were conducted on male Wistar rats divided into three groups: 1. Control, 2. fed HFD and 3. fed HFD and treated with metformin. The animals were infused with a [U-13C]palmitate to measure fractional lipid synthesis rate. This allowed for the calculation of fractional synthesis rate of signaling lipids (FSR) through the estimation of their isotopic enrichment. Liver ceramide (Cer), diacylglycerol (DAG) and acyl-carnitine concentration and enrichment were analyzed by LC/MS/MS. The content of proteins involved in lipid metabolism and insulin signaling were analyzed by Western Blot. HFD treatment increased the content and FSR of DAG and Cer in the liver which was accompanied by systemic insulin resistance and inhibition of hepatic insulin signaling pathway under insulin stimulation. Metformin treatment ameliorated systemic insulin resistance and augmented the hepatic insulin signaling cascade. It reduced both the concentration and FSR of Cer, DAG, and increased acyl-carnitine content and the expression of mitochondrial markers. We postulate, that in liver, the insulin sensitizing effect of metformin depends on augmentation of mitochondrial ß-oxidation, which protects from hepatic accumulation of both the Cer and DAG and preserves insulin sensitivity under HFD consumption. Moreover, we showed that hepatic content of Cer and DAG corresponds with their respective FSR.

Forsskamide, a new ceramide from aerial parts of Forsskaolea tenacissima Linn.

Although the various folk medicine uses and the biological activity of Forsskaolea tenacissima L., few chemical constituents of this plant have been reported, this provoked us to make our study. Forsskamide, a new ceramide was isolated from aerial parts of F. tenacissima L. (Urticaceae). The chemical structure was established by different spectroscopic methods (1H, 13C-NMR, HMBC, HSQC, ROESY, FAB-MS and HR-FAB-MS). Forsskamide showed a moderate cytotoxic activity by (MTT) method against human colorectal carcinoma cell line (HCT-116) with IC50 33.25 µM in comparison with 5-fluorouracil IC50 26.42 µM. While, it did not show any activity against human hepatocarcinaoma cell line (HepG-2).

Soft coral Cespitularia stolonifera: New cytotoxic ceramides and gastroprotective activity.

In the present study, a new ceramide, namely 2S, 3R-4E, 8E-2-(heptadecanoylamino)-heptadeca-4, 8-diene-1, 3-diol (1), along with four known steroids, including 24-methylcholesta-5, 24(28)-diene-3ß-ol (2), 24-methylcholesta-5, 24(28)-diene-3ß-acetate (3), 4-methyl-24-methylcholesta-22-ene-3-ol (4), and cholesterol, was isolated and characterized from CH2Cl2/MeOH extract of Cespitularia stolonifera. A new acetate derivative of compound 1, termed 2S, 3R-4E, 8E-2-(heptadecanoylamino)-heptadeca-4, 8-diene-1, 3-diacetate (1a), was also prepared in the present study. All the structures were established on the basis of modern spectroscopic techniques, including FT-IR, 1D, 2D-NMR, HRESI-MS, and GC-MS, in addition of chemical methods. (-)-Alloaromadendren, ledane, (1)-alloaromadendren oxide, isoaromadendrene epoxide and (-)-caryophellen oxide were identified from the n-hexane fraction using GC-MS. The extract and the two ceramides (1) and (1a) exhibited significant cytotoxic activity against lung cancer A549 cells, while the extract and the two steroids (2) and (3) exhibited significant cytotoxic activity against breast cancer MCF-7 cells. The CH2Cl2/MeOH extract exhibited significant antiulcer activity in both ethanol and acetic acid induced ulcer models in rats, as evidenced by histopathological, histochemical, and biochemical examinations.

Linear ion-trap MSn with high-resolution MS reveals structural diversity of 1-O-acylceramide family in mouse epidermis.

1-O-acylceramide is a new class of epidermal cer-amide (Cer) found in humans and mice. Here, we report an ESI linear ion-trap (LIT) multiple-stage MS (MSn) approach with high resolution toward structural characterization of this lipid family isolated from mice. Molecular species desorbed as the [M + H]+ ions were subjected to LIT MS2 to yield predominately the [M + H - H2O]+ ions, followed by MS3 to cleave the 1-O-acyl residue to yield the [M + H - H2O - (1-O-FA)]+ ions. The structures of the N-acyl chain and long-chain base (LCB) of the molecule were determined by MS4 on [M + H - H2O - (1-O-FA)]+ ions that yielded multiple sets of specific ions. Using this approach, isomers varied in the 1-O-acyl (from 14:0- to 30:0-O-acyl) and N-acyl chains (from 14:0- to 34:1-N-acyl) with 18:1-sphingosine as the major LCB were found for the entire family. Minor isomers consisting of 16:1-, 17:1-, 18:2-, and 19:1-sphingosine LCBs with odd fatty acyl chain or with monounsaturated N- or O-fatty acyl substituents were also identified. An estimation of more than 700 1-O-acylceramide species, largely isobaric isomers, are present, underscoring the complexity of this Cer family.

Production of Rare Phyto-Ceramides from Abundant Food Plant Residues.

Ceramides (Cers) are major components of the outermost layer of the skin, the stratum corneum, and play a crucial role in permeability barrier functions. Alterations in Cer composition causing skin diseases are compensated with semisynthetic skin-identical Cers. Plants constitute new resources for Cer production as they contain glucosylceramides (GluCers) as major components. GluCers were purified from industrial waste plant materials, apple pomace (Malus domestica), wheat germs (Triticum sp.), and coffee grounds (Coffea sp.), with GluCer contents of 28.9 mg, 33.7 mg, and 4.4 mg per 100 g of plant material. Forty-five species of GluCers (1-45) were identified with different sphingoid bases, saturated or monounsaturated α-hydroxy fatty acids (C15-28), and ß-glucose as polar headgroup. Three main GluCers were hydrolyzed by a recombinant human glucocerebrosidase to produce phyto-Cers (46-48). These studies showed that rare and expensive phyto-Cers can be obtained from industrial food plant residues.