Hydrogen peroxide modification affects the structure and physicochemical properties of dietary fibers from white turnip (Brassica Rapa L.).

Turnip (Brassica rapa L.) is widely consumed as a vegetable and traditional Chinese medicine with high dietary fiber content. Soluble dietary fiber (SDF) and insoluble dietary fiber (IDF) were obtained from white turnips, and the IDF was modified with alkaline hydrogen peroxide to obtain modified IDF (MIDF) and modified SDF (MSDF). The compositional, structural, and functional properties of the four samples were investigated. After modification, the modified dietary fibers (MDFs) showed smaller particle sizes and lower contents of pectin and polyphenol than those of unmodified dietary fibers (DFs) The results of scanning electron microscopy (SEM), Fourier transformed infrared (FT-IR) spectroscopy, X-ray diffraction (XRD) and differential scanning calorimetry (DSC) showed that compared to the DFs, the MDFs were smaller and had more exposed hydroxyl groups. Analysis of the microrheological behaviors showed that the MDFs had higher viscosity than that of the DFs, with a looser structure for the MSDF and a stable structure for the MIDF. Therefore, due to structural changes, the physical and functional properties of the MDFs were improved compared to those of the unmodified DFs. Pearson correlation analysis showed that the particle size was positively correlated with the pectin content. The water holding capacity (WHC), oil adsorption capacity (OAC) and water swelling capacity (WSC) showed positive correlations with each other. This work indicated that white turnip could be a potential new source of DFs, which presented desirable functional properties after modification.

Evaluation of Antioxidant and Antibacterial Activities, Cytotoxicity of Acacia seyal Del Bark Extracts and Isolated Compounds.

Water extract of Acacia seyal bark is used traditionally by the population in Djibouti for its anti-infectious activity. The evaluation of in vitro antibacterial, antioxidant activities and cytotoxicity as well as chemical characterization of Acacia seyal bark water and methanolic extracts were presented. The water extract has a toxicity against the MRC-5 cells at 256 µg/mL while the methanolic extract has a weak toxicity at the same concentration. The methanolic extract has a strong antioxidant activity with half maximal inhibitory concentration (IC50) of 150 ± 2.2 µg/mL using 1-diphenyl-2-picrylhydrazyl (DPPH) and IC50 of 27 ± 1.3 µg/mL using 2,2'-azino-bis 3-ethylbenzthiazoline-6-sulphonic acid (ABTS) radical methods. For ferric reducing/antioxidant power (FRAP) assay, the result is 45.74 ± 5.96 µg Vitamin C Equivalent (VCE)/g of dry weight (DW). The precipitation of tannins from methanol crude extract decreases the MIC from 64 µg/mL to 32 µg/mL against Staphylococcus aureus and Corynebacterium urealyticum. However, the antioxidant activity is higher before tannins precipitation than after (IC50 = 150 µg/mL for methanolic crude extract and 250 µg/mL after tannins precipitation determined by DPPH method). By matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, the results showed that the condensed tannins consist of two types of catechin and gallocatechin-based oligomers. The fractionation led to the identification of three pure compounds: two flavanols catechin and epicatechin; one triterpene as lupeol; and a mixture of three steroids and one fatty acid: campesterol, stigmasterol, clionasterol, and oleamide.

Adsorptive removal of cholesterol by biodegradable zein-graft-ß-cyclodextrin film.

A novel bio-based zein-graft-ß-cyclodextrin film was synthesized for cholesterol adsorption at room temperature. Immobilization of ß-cyclodextrin (ß-CD) to zein was achieved by the Maillard reaction and the zein-graft-ß-CD powders showed higher glass transition temperature and higher solubility in water. Zein-graft-ß-CD films, prepared by solvent evaporation casting, showed excellent capacity for cholesterol adsorption. The F1:1 has the best adsorption properties, and the maximum adsorption capacity can reach 5.70 ± 0.56 mg cholesterol/g film. The adsorption cholesterol mechanism of zein-graft-ß-CD film was correspond to the pseudo-first order kinetic model. In addition, the zein-graft-ß-CD film retained an adsorption capacity of 3.10 ± 0.89 mg cholesterol/g film after three reuses. Using composite enzyme (1 mg/mL papain and 1 mg/mL amylase), the film (F1:1) degradation reached 98.81% in 1 week. These results suggest that zein-graft-ß-CD film has potential as a nature-based adsorbent for cholesterol adsorption and could be used in the food industry.

Molecularly imprinted cryogel beads for cholesterol removal from milk samples.

This study presents the development of a cholesterol-selective adsorbent that can be easily produced for the highly efficient removal of cholesterol from milk. A fundamental affinity separation technology which was combined with the specific recognition property of molecular imprinting with a high flow rate and the resulting cryogel was used to separate cholesterol separation from milk samples. The proposed material offers a reasonable pore size and structure, high surface area, and mechanical and chemical stability. To separation the cholesterol from milk, poly(2-hyroxyethyl methacrylate-N-methacryloyl-l-tryptophan methylester) cryogel beads were prepared using a functional monomer that allowed the formation of cholesterol-selective binding sites and enhanced the selective removal of cholesterol from milk. Characterization studies of the cholesterol-imprinted cryogel beads (CHO-MIPs) were carried out by attenuated total reflectance-Fourier transform infrared spectroscopy, scanning electron microscopy, elemental analysis, water-uptake tests and surface area measurements. The interactions between CHO-MIP and cholesterol were investigated and the factors affecting the adsorption of cholesterol were determined to find optimum conditions. Reusability as a measure of the continuity of the prepared CHO-MIPs was also investigated. The selectivity of the CHO-MIP beads was determined by using competing molecules (estradiol and progesterone), which are cholesterol analogues. The experimental data showed that the specific areas of the CHO-MIP and non-imprinted (NIP) cryogel beads were 17.6 and 14.7 m2/g, respectively. The CHO-MIP cryogel beads were 4.77 and 2.76 fold more selective for cholesterol compared to estradiol and progesterone respectively. The cholesterol adsorption capacity of the CHO-MIP beads was 288.72 mg/g when the cholesterol concentration in solution was 3.0 mg/mL. After eight adsorption-desorption cycles, the adsorption capacity of the CHO-MIP beads decreased by 9.21 %. The Langmuir-Freundlich isotherm model was well fitted as compare to Langmuir and Freundlich isotherms. The obtained kinetics data showed that a pseudo-second order mechanism was predominant for the CHO-MIP cryogel bead adsorption.

Real-time cholesterol sorting in Plasmodium falciparum-erythrocytes as revealed by 3D label-free imaging.

Cholesterol, a necessary component of animal cell membranes, is also needed by the lethal human malaria parasite Plasmodium falciparum. Because P. falciparum lacks a cholesterol synthesis pathway and malaria patients have low blood cholesterol, we speculated that it scavenges cholesterol from them in some way. We used time-lapse holotomographic microscopy to observe cholesterol transport in live P. falciparum parasites and structurally investigate erythrocyte membranes, both during and after P. falciparum invasion of human erythrocytes. After P. falciparum initially acquired free cholesterol or inner erythrocytic membrane-derived cholesterol, we observed budding lipid membranes elongating into the cytosol and/or membrane segments migrating there and eventually fusing with the parasite membranes, presumably at the parasitophorous vacuole membrane (PVM). Finally, the cholesterol-containing segments were seen to surround the parasite nucleus. Our imaging data suggest that a novel membrane transport system operates in the cytosol of P. falciparum-infected erythrocytes as a cholesterol import system, likely between the PVM and the erythrocyte membrane, and that this transportation process occurs during the live erythrocyte stages of P. falciparum.

Analysis of cholesterol in mouse brain by HPLC with UV detection.

We describe a sensitive high performance liquid chromatography (HPLC)-based method for the determination of cholesterol in brain tissue. The method does not require the derivatization of the analyte and uses separation and quantification by reversed-phase HPLC coupled to UV detection. Lipids were methanol/chloroform extracted following the method of Bligh and Dyer, and separated using isopropanol/acetonitrile/water (60/30/10, v/v/v) as mobile phase. We observed lineal detection in a wide range of concentrations, from 62.5 to 2000 ng/µL, and were able to detect a significant increase in the brain cholesterol levels between postnatal days 2 and 10 in C57BL6 mice. Based on our validation parameters, we consider this analytical method a useful tool to assess free cholesterol in rodent brain samples and cell cultures.

Evaluation of biological activities, and exploration on mechanism of action of matrine-cholesterol derivatives.

To develop new potential pesticides, a series of matrine-cholesterol derivatives were prepared by modifications of two non-food bioactive products matrine and cholesterol. Two N-phenylsulfonylmatrinic esters (5i and 5j) showed the most potent insecticidal activity against Mythimna separata Walker. Two N-benzylmatrinic esters (5e and 5g) exhibited the most promising aphicidal activity against Aphis citricola Van der Goot. Especially compound 5e showed good control effects in the greenhouse against A. citricola. Some interesting results of their structure-activity relationships were also observed. By reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time polymerase chain reaction (qRT-PCR) analysis of HMG-CoA reductase in apterous adults of A. citricola, it demonstrated that matrine and cholesterol may be the HMG-CoA reductase inhibitors, and the hydroxyl of cholesterol or the lactam ring of matrine may be important for acting with HMG-CoA reductase in A. citricola.

Influence of Oxygen on Function and Cholesterol Composition of Murine Bone Marrow-Derived Neutrophils.

During inflammation and infection, invading pathogens as well as infiltrating neutrophils locally consume oxygen and reduce the present oxygen level. Since oxygen is an elementary component of the microenvironment required for cell activity and alters multiple cellular functions, it is important to study neutrophil functionality and phenotype at characteristic pathophysiological oxygen levels that reflect the hypoxic phenotype during infection and inflammation. Here, we describe methods to study murine neutrophils under hypoxic compared to normoxic conditions, including analysis of cholesterol content as a key lipid involved in biological functions.

Amphipathic molecules modulate PIEZO1 activity.

PIEZO proteins are large eukaryotic mechanically-gated channels that function as homotrimers. The basic PIEZO1 structure has been elucidated by CryoEM and it assembles into a protein-lipid dome. A curved lipid region allows for the transition to the lipid bilayer from the dome (footprint). Gating PIEZO1 is mediated by bilayer tension that induces an area change in the lipid dome. The footprint region is thought to be energetically important for changes in lateral tension. Amphipathic molecules can modulate channel function beyond the intrinsic gating properties of PIEZO1. As a result, molecules that modify lipid properties within the lipid-channel complex (footprint and dome) will profoundly affect channel kinetics. In this review, we summarize the effects some amphipathic molecules have on the lipid bilayer and PIEZO1 function. PIEZO1 has three states, closed, open and inactivated and amphipathic molecules influence these transitions. The amphipathic peptide, GsMTx4, inhibits the closed to open transition. While saturated fatty acids also prevent PIEZO1 gating, the effect is mediated by stiffening the lipids, presumably in both the dome and footprint region. Polyunsaturated fatty acids can increase disorder within the lipid-protein complex affecting channel kinetics. PIEZO1 can also form higher-ordered structures that confers new kinetic properties associated with clustered channels. Cholesterol-rich domains house PIEZO1 channels, and depletion of cholesterol causes a breakdown of those domains with changes to channel kinetics and channel diffusion. These examples underscore the complex effects lipophilic molecules can have on the PIEZO1 lipid dome structure and thus on the mechanical response of the cell.

Cholesterol, a powerful 13C isotopic biomarker.

Cholesterol is related to many health diseases and is considered as a metabolic disorder biomarker. This compound, present in all food products of animal origin, can also be used as food authentication biomarker. In this work and for the first time, positional 13C isotope contents were determined for such a high molecular weight compound. This was possible by means of NMR using adiabatic refocused INEPT. In order to test the potential of this approach for discrimination, hen eggs from different origins were collected. Quantitative extraction of egg yolk cholesterol was optimized, and partial reduced molar fractions of its different 13C isotopomers were used as predictors in discriminant analysis. Compared with the global 13C isotopic composition determined using isotope ratio monitoring by Mass Spectrometry, the relative content of cholesterol 13C isotopomers added valuable power to sample classifications according to their origins. This study paves the way to isotopomics of other steroids and similar molecular weight compounds.

CuO nanoparticles derived from metal-organic gel with excellent electrocatalytic and peroxidase-mimicking activities for glucose and cholesterol detection.

A simple and efficient strategy was developed to fabricate CuO nanoparticles (CuO-NPs) with high surface area by the direct pyrolysis of a metal-organic gel (MOG) precursor for constructing versatile catalytic interfaces. Unexpectedly, the obtained CuO-NPs exhibited excellent electrocatalytic activity for glucose (Glu) oxidation reaction. The linear range of glucose was from 5 µM to 600 with the detection limit of 0.59 µM. Additionally, the CuO-NPs showed distinguished intrinsic peroxidase-mimicking activities, which can be further used as biomimetic nanozymes for sensitively and rapidly detecting cholesterol. A good linearity of cholesterol was performed in the range from 1 µM to 15 µM with the detection limit of 0.43 µM. The as-prepared CuO-NPs could provide a versatile catalytic platform for the application of electrochemical sensors and biomimetic enzyme catalytic systems. This study proved the high potential of MOG-derived nanostructured transition metal oxides (TMOs) with multiple complex functions.

Extraction of phospholipid-rich fractions from egg yolk and development of liposomes entrapping a dietary polyphenol with neuroactive potential.

A new protocol to obtain egg yolk phospholipids in ethanol is presented. Rutin-phospholipids nanoliposomes were prepared and characterized. The procedure takes advantage of the different solubility of egg yolk lipids in ethanol and acetone at low temperature, to efficiently obtain a phospholipid-rich fraction of high purity degree. The phospholipid content in the final fraction is 208.65 ± 26.46 µmol/g fresh egg yolk (16%), accounting for ca. 96% of the extract's dry weight. The phospholipid-rich fraction contains cholesterol (0.069-0.082 cholesterol/phospholipid molar ratio), and vestigial amounts of lutein and zeaxanthin (89.24 ± 9.76 and 14.9 ± 2.16 ng/g of fresh egg yolk, respectively). Saturated fatty acids dominate the extracted phospholipids (50% of egg's total yolk phospholipids), the levels of monounsaturated ranging from 20 to 25%, and polyunsaturated up to 35%. Rutin-liposomes, prepared with phospholipid-rich fraction, presented mean diameter <140 nm, negative surface charge (Zeta potential ~ -13 mV), and entrapment efficiency of rutin up to 87%. In human neuroblastoma cell line SH-SY5Y, rutin-liposomes (lipid 25 µM + rutin 16.7 µM) attenuated glutamate-induced cytotoxicity, in part by reducing the formation of intracellular reactive species, pointing to their potential application as new functional neuroprotective agents.

Nanodot-Directed Formation of Plasmonic-Fluorescent Nanohybrids toward Dual Optical Detection of Glucose and Cholesterol via Hydrogen Peroxide Sensing.

Hybrid nanoparticles (NPs) have emerged as an important class of nanomaterials owing to their integrated enhanced properties and functionality. In this study, we have developed an effective nanodot templating strategy for the in situ formation of surfactant-free nanohybrids with unique plasmonic-fluorescent properties. A bright photoluminescent biodot synthesized from serine and histamine biomolecular precursors (Ser-Hist dot) was first engineered to have rich functional groups on the nanosurface capable of anchoring Ag+ ions via electrostatic interaction. Upon UV irradiation, free electrons could transfer from the photoexcited Ser-Hist dot to the Ag+ ions, facilitating the in situ growth of AgNPs. The resulting nanohybrid system (Bio@AgNPs) exhibits distinct characteristic surface plasmon resonance absorbance and highly quenched PL intensity due to the inner filter effect. Furthermore, the Bio@AgNP nanohybrid retains its redox capability, enabling hydrogen peroxide sensing via AgNP etching, which in turn empowers a dual colorimetric and fluorescent detection of glucose and cholesterol in complex biological samples (i.e., synthetic urine and human plasma) with high selectivity and sensitivity. This finding reveals a new effective and facile method for the preparation of highly functional hybrid nanomaterials for dual-mode detection of hydrogen peroxide-producing species and/or reactions.

Optical Nanosensing of Lipid Accumulation due to Enzyme Inhibition in Live Cells.

Drugs that influence enzymes of lipid metabolism can cause pathological accumulation of lipids in animal cells. Here, gold nanoparticles, acting as nanosensors that deliver surface-enhanced Raman scattering (SERS) spectra from living cells provide molecular evidence of lipid accumulation in lysosomes after treatment of cultured cells with the three tricyclic antidepressants (TCA) desipramine, amitryptiline, and imipramine. The vibrational spectra elucidate to great detail and with very high sensitivity the composition of the drug-induced lipid accumulations, also observed in fixed samples by electron microscopy and X-ray nanotomography. The nanoprobes show that mostly sphingomyelin is accumulated in the lysosomes but also other lipids, in particular, cholesterol. The observation of sphingomyelin accumulation supports the impairment of the enzyme acid sphingomyelinase. The SERS data were analyzed by random forest based approaches, in particular, by minimal depth variable selection and surrogate minimal depth (SMD), shown here to be particularly useful machine learning tools for the analysis of the lipid signals that contribute only weakly to SERS spectra of cells. SMD is used for the identification of molecular colocalization and interactions of the drug molecules with lipid membranes and for discriminating between the biochemical effects of the three different TCA molecules, in agreement with their different activity. The spectra also indicate that the protein composition is significantly changed in cells treated with the drugs.

Measurement of Bile Acids as a Marker of the Functionality of iPSC-Derived Hepatocytes.

During the process of differentiation from induced pluripotent stem cells (iPSCs) to hepatocyte-like cells it is crucial to monitor the functionality of cells, in order to avoid an overestimation of the level of maturation. To this end, we propose bile acid profiling as a novel approach which is useful in determining the maturation of hepatocyte-like cells. The main advantages of the method are the simplicity and rapidity of test (i.e., single-step sample preparation followed by 3.5-min analysis), as well as the possibility to localize possible enzyme deficiencies by quantifying the accumulation of specific intermediates involved in the synthetic pathways.

Analyses of Cholesterol Metabolites of Optic Nerve Using GC-MS Methods.

Gas chromatography-mass spectrometry (GC-MS) is considered the gold standard for analyzing and quantifying the presence of biological compounds in tissue samples due to its high sensitivity, peak resolution, and reproducibility. In this chapter, we describe a step-by-step modified Bligh and Dyer protocol for lipid extraction from the optic nerve tissue and a procedure for GC-MS analyses of the lipid extract. These protocols are based on our experience and can be modified depending on samples and compounds of interest.

Nonglucuronidated Ezetimibe Disrupts CD13- and CD64-Coassembly in Membrane Microdomains and Decreases Cellular Cholesterol Content in Human Monocytes/Macrophages.

Ezetimibe (EZE) and glucuronidated EZE (EZE-Glu) differentially target Niemann-Pick C1-like 1 (NPC1L1) and CD13 (aminopeptidase-N) to inhibit intestinal cholesterol absorption and cholesterol processing in other cells, although the precise molecular mechanisms are not fully elucidated. Cellular effects of EZE, EZE-Glu, and the low-absorbable EZE-analogue S6130 were investigated on human monocyte-derived macrophages upon loading with atherogenic lipoproteins. EZE and S6130, but not EZE-Glu disturbed the colocalization of CD13 and its coreceptor CD64 (Fcγ receptor I) in membrane microdomains, and decreased the presence of both receptors in detergent-resistant membrane fractions. Biotinylated cholesterol absorption inhibitor C-5 (i.e., derivative of EZE) was rapidly internalized to perinuclear tubular structures of cells, resembling endoplasmic reticulum (ER), but CD13 was detected on extracellular sites of the plasma membrane and endolysosomal vesicles. Administration of EZE, but not of EZE-Glu or S6130, was associated with decreased cellular cholesteryl ester content, indicating the sterol-O acyltransferase 1 (SOAT1)-inhibition by EZE. Furthermore, EZE decreased the expression of molecules involved in cholesterol uptake and synthesis, in parallel with increased apolipoprotein A-I-mediated cholesterol efflux and upregulation of efflux-effectors. However, NPC1L1 the other claimed molecular target of EZE, was not detected in macrophages, thereby excluding this protein as target for EZE in macrophages. Thus, EZE is very likely a CD13-linked microdomain-disruptor and SOAT1-inhibitor in macrophages leading to in vitro anti-atherosclerotic effects through a decrease of net cellular cholesterol content. © 2019 International Society for Advancement of Cytometry.

Separation and purification of lanosterol, dihydrolanosterol, and cholesterol from lanolin by high-performance counter-current chromatography dual-mode elution method.

Lanosterol is a potential drug for cataracts treatment, which can reverse the aggregation of intracrystalline proteins. The low concentration in lanolin calls for high-performance separation methods. In this study, a counter-current chromatography dual-mode elution method was developed for the first time to separate and purify lanosterol from hexane extract of lanolin after saponification, in which the column was first eluted with the lower phase as mobile phase in head-to-tail mode, followed by the upper phase in the tail-to-head mode. High purity of lanosterol, dihydrolanosterol, and cholesterol can be obtained simultaneously. A solvent system composed of n-heptane/acetonitrile/ethyl acetate (5:5:1, v/v/v) was selected and optimized via partition coefficient determination. Compounds such as 111 mg lanosterol, 84 mg dihydrolanosterol, and 183 mg cholesterol with high purity of 99.77, 95.71, and 91.43%, respectively, analyzed by high-performance liquid chromatography were obtained within 80 min from 700 mg crude extract from 1.78 g lanolin. The method was also used to improve the purity of commercial lanosterol product from 66.97 to above 99%. Counter-current chromatography could serve as a potential and powerful technique for commercial production of highly pure lanosterol.