Cholic Acid Sodium Salt Research Articles

Expression and Purification of the Full Length and N-Terminal Truncated Variants of Insect CYP6Z2 in the Cytosol of Escherichia Coli for Potential 3D Experimental Studies

Cytochrome P450 enzymes (P450s) offer innate resistance defence for malaria vectors against the insecticides permitted by WHO to be used in vector control tools. P450s can detoxify broad substrates and simultaneously metabolise them, thus the availability of experimental three-dimensional structures of these key insecticide detoxifiers is vital to improving our knowledge of their enzyme activities. Despite the importance of this family of proteins in insecticide resistance, there are no available experimental three-dimensional structures of insect P450 yet. For this investigation, a carboxy-terminal Histidine-tagged recombinant CYP6Z2 was heterologously expressed in E. coli to generate a soluble holoprotein suitable for an experimental three-dimensional structure. The expressed enzyme was purified from the cytosol of E. coli via the combination of various purification techniques and cholic acid sodium salt. Two truncated N-terminal signal peptides: short deletion of 11 amino acids and long deletion of 23 amino acids of the hydrophobic domain, were created to prevent aggregation, improve solubility, and facilitate crystallisation. The CYP6Z2 (full length) produced a holoprotein with a P450 protein concentration of 0.60 nmol/mL, whereas the two truncated CYP6Z2 isoforms produced only the inactive species with no peak at 450 nm. We conclude that the hydrophobic signal peptide region of the insect Cytochrome P450s seems sensitive and indispensable to ensuring 3-D folding and stability.

Aqueous electrophoretic deposition of drugs using bile acids as solubilizing, charging and film-forming agents

A new electrophoretic deposition (EPD) technique is reported for the fabrication of composite coatings containing drugs, which have low solubility in water. We present a conceptually new approach, which is based on the use of bile salts (BS) as solubilizing, charging and film forming agents. Composite films are obtained using tetracycline and ibuprofen as model drugs with commercial BS, such as cholic acid sodium salt and deoxycholic acid sodium salt. The suggested mechanism involves the solubilization of drugs in BS solutions, followed by the formation of mixed charged micelles, containing drugs and charged BS, which allow electrophoretic transport of the drugs to the electrode surface. The EPD mechanism involves pH decrease at the anode surface, protonation of carboxylic groups of BS and formation of water insoluble films. The approach can be used for deposition of other functional organic molecules and inorganic nanoparticles.

EXPLOITING THE VERSATILITY OF CHOLESTEROL IN NANOPARTICLES FORMULATION

The biocompatibility of polymers, lipids and surfactants used to formulate is crucial for the safe and sustainable development of nanocarriers (nanoparticles, liposomes, micelles, and other nanocarriers). In this study, Cholesterol (Chol), a typical biocompatible component of liposomal systems, was formulated in Chol-based solid nanoparticles (NPs) stabilized by the action of surfactant and without the help of any other formulative component. Parameters as type (Solutol HS 15, cholic acid sodium salt, poly vinyl alcohol and Pluronic-F68), concentration (0.2; 0.5 and 1% w/v) of surfactant and working temperature (r.t. and 45°C) were optimized and all samples characterized in terms of size, zeta potential, composition, thermal behavior and structure. Results demonstrated that only Pluronic-F68 (0.5% w/v) favors the organization of Chol chains in structured NPs with mean diameter less than 400nm. Moreover, we demonstrated the pivotal role of working temperature on surfactant aggregation state/architecture/stability of Chol-based nanoparticles. At room temperature, Pluronic-F68 exists in solution as individual coils. In this condition, nanoprecipitation of Chol formed the less stable NPs with a 14±3% (w/w) of Pluronic-F68 prevalently on surface (NP-Chol/0.5). On the contrary, working near the critical micelle temperature (CMT) of surfactant (45°C), Chol precipitates with Pluronic-F68 (9±5% w/w) in a compact stable matricial structure (NP-Chol/0.5-45). In vitro studies highlight the low toxicity and the affinity of NP-Chol/0.5-45 for neuronal cells suggesting their potential applicability in pathologies with a demonstrated alteration of neuronal plasticity and synaptic communication (i.e. Huntington⿿s disease).

Reduced Graphene Oxide Nanosheets Functionalized with Bile Salts as Support for Electrochemical Catalysts

Graphene nanosheets functionalized with bile salts (G-bile salts) are obtained through chemical reduction of exfoliated graphite oxide in the presence of deoxycholic acid sodium salt, taurodeoxycholic acid sodium salt, or cholic acid sodium salt. Due to the “facial amphiphilic” structure of bile salt molecules, the G-bile salt composites exhibit excellent dispersibility in water. In addition, G-bile salts decorated with palladium (Pd) nanoparticles (Pd-G-bile salts) are prepared via a facile co-reduction process. The use of the Pd-G-bile salt composites as electrochemical catalysts for formic acid oxidation reaction is demonstrated. Better utilization has been achieved for Pd catalysts dispersed on G-bile salts than those prepared without bile salts.

Thermodynamic Origin of Selective Binding of β-Cyclodextrin Derivatives with Chiral Chromophoric Substituents toward Steroids

Two β-cyclodextrin derivatives with chiral chromophoric substituents, that is, L- (1) and D-tyrosine-modified β-cyclodextrin (2), were synthesized and fully characterized. Their inclusion modes, binding abilities, and molecular selectivities with four steroid guests, that is, cholic acid sodium salt (CA), deoxycholic acid sodium salt (DCA), glycochoic acid sodium salt (GCA), and taurocholic acid sodium salt (TCA), were investigated by the circular dichroism, 2D NMR, and isothermal titration microcalorimetry (ITC). The results obtained from the circular dichroism and 2D NMR showed that two hosts adopted the different binding geometry, and these differences subsequently resulted in the significant differences of molecular binding abilities and selectivities. As compared with native β-cyclodextrin and tryptophan-modified β-cyclodextrin, host 2 showed the enhanced binding abilities for CA and DCA but the decreased binding abilities for GCA and TCA; however, host 1 showed the decreased binding abilities for all four bile salts. The best guest selectivity and the best host selectivity were K(S)(2-DCA)/K(S)(2-TCA) = 12.6 and K(S)(2-CA)/K(S)(1-CA) = 10, respectively, both exhibiting great enhancement as compared with the corresponding values of the previously reported L- and D-tryptophan-modified β-cyclodextrins. Thermodynamically, it was the favorable enthalpic gain that led to the high guest selectivity and host selectivity.

Novel Permethylated β-Cyclodextrin Derivatives Appended with Chromophores as Efficient Fluorescent Sensors for the Molecular Recognition of Bile Salts

Two novel permethylated beta-cyclodextrin (PM-beta-CD) derivatives, i.e., 6I-O-(1-naphtholxy)-2I,31-di-O-methylhexakis(2II-VII,3II-VII,6II-VII-tri-O-methyl)-beta-cyclodextrin (1) and 6I-O-(8-hydroxyquinoline)-2I,31-di-O-methylhexakis(2II-VII,3II-VII,6II-VII- tri-O-methyl)-beta-cyclodextrin (2), were synthesized in satisfactory yields, and their inclusion modes, complex-induced fluorescent behaviors, binding ability, and selectivity for bile salts of biological relevance (cholic acid sodium salt, CA; deoxycholic acid sodium salt, DCA; glycochoic acid sodium salt, GCA; taurocholic acid sodium salt, TCA) were investigated by the circular dichroism, 2D NMR, steady-state, and time-resolved fluorescent spectra. The results obtained from induced circular dichroism and ROESY spectra show that the chromophore groups of 1 and 2 reside in the central cavity of PM-beta-CD, and are expelled to the region of narrow torus rim upon complexation with bile guests, which presents the binding mode of cooperative inclusion. The transfer of the chromophore groups from the central cavity to the more hydrophobic torus rim leads to the remarkable increase of fluorescent intensities and longer fluorescent lifetimes of hosts 1 and 2 upon gradual addition of bile salts, which is importantly distinct from the molecular recognition of the chromophore-modified beta-CD species with bile salts. Interestingly, hosts 1 and 2 present much stronger binding ability for bile guests than PM-beta-CD. Differing from native beta-CD, all the PM-beta-CDs are more prone to include bile salts with longer tails, such as GCA and TCA. Their corresponding binding ability and molecular selectivity are closely discussed from the viewpoints of difference of cavity size/shape between beta-CD and PM-beta-CD, effect of substituent groups, and structures of bile guests, respectively.

Synthesis and characterization of spinelic ferrites obtained from coordination compounds as precursors

Cobalt ferrite and magnetite nanoparticles were obtained by “the complexation method” belonging to soft chemistry, in which cholic acid sodium salt acts as complexation agent. The precursors were characterized by IR, UV–vis spectra and magnetic measurements. X-ray diffraction spectra and transmission electron microscopy proved the nanocrystalline structure of the spinelic ferrites.

Formation of Giant Needle‐Like Polycation‐Bile Acid Complexes

AbstractThe complexation of bile acids with various solvated polycations was studied. A one‐to‐one complex was precipitated when an aqueous solution of cholic acid sodium salt (CA) was mixed with aqueous solutions of 3,3‐ionene and grew to form crystals with needle‐like morphology, 3 millimeters in length. Hydrogen bonding of hydroxyls at the steroid face and the spacing between cationic sites of polycations were crucial for the formation of the giant needle.Crossed polarizing microscopic photograph of the complex composed of cholic acid sodium salt and 3,3‐ionene.magnified imageCrossed polarizing microscopic photograph of the complex composed of cholic acid sodium salt and 3,3‐ionene.

Lipase degradation of Dynasan 114 and 116 solid lipid nanoparticles (SLN)—effect of surfactants, storage time and crystallinity

In vivo drug release from solid lipid nanoparticles (SLN) takes place by diffusion and degradation of the lipid matrix. SLN with different degree of crystallinity were prepared to study the effect of crystallinity on the degradation velocity. These SLN were produced by using glycerides with different length of fatty acid chains and known differences in crystallisation velocity (Dynasan 114 and 116), and using stabilisers interfering differently with the crystallisation process of the lipid matrix (cholic acid sodium salt (NaCh), Poloxamer 407 (Plx 407)). NaCh disturbs the crystallisation process, Poloxamer shows little interference. The particles were characterised by photon correlation spectroscopy (PCS) and differential scanning calorimetry (DSC), degradation velocity was determined directly after production and during storage up to 4 weeks under different storage conditions using an especially developed assay based on the NEFA Test kit. After production, SLN with a lower crystallinity matrix (Dynasan 114 and 116, NaCh) degraded faster than higher crystalline particles (all SLN with Plx 407), and showed a decrease in degradation velocity with increasing crystallinity during storage. Fast crystallising particles made from Dynasan 116 stabilised with the non-interfering Plx 407 showed no change in the degradation velocity during storage. SLN produced with a higher crystalline lipid in combination with the crystallisation-disturbing NaCh (Dynasan 116, NaCh) required a ‘ripening time’ to reach sufficient crystallinity.

Enzymatic Degradation of Dynasan 114 SLN – Effect of Surfactants and Particle Size

The degradation velocity of solid lipid nanoparticles (SLN) is – apart from drug diffusion – an important parameter determining drug release in vivo. To assess the effect of stabilizers systematically, Dynasan 114 SLN were produced with ionic surfactants (e.g. cholic acid sodium salt (NaCh), sodium dodecyl sulfate (SDS), cetylpyridiniumchloride (CPC)) and steric stabilizers (Tween 80, Poloxamer 188, 407 and Poloxamine 908) including a mixture of cholic acid sodium salt and Poloxamer 407. In addition, the size effects were investigated. The degradation velocity was measured using an in vitro lipase assay. SLN stabilized with lecithin and NaCh showed the fastest, Tween 80 the intermediate and the high molecular weight Poloxamer 407 the slowest degradation. Size effects were less pronounced for fast degrading particles (e.g. those stabilized with NaCh). No difference in the size range of 180–300-nm was observed, but a distinctly slower degradation of 800-nm SLN could be detected. For slowly degrading particles, more pronounced size effects were found. Size effects are more difficult to assess when the PCS diameters are similar, but small fractions of micrometer particles are present, besides the nanometer bulk population. The measured FFA formation is then a superposition of particles degrading at different speeds due to differences in the shape of the size distribution. Admixing of Poloxamer to NaCh had no delaying effect on the degradation of the Dynasan 114 SLN, indicating an influence of the nature of the lipid matrix that is affecting the stabilizers affinity to and anchoring onto the SLN surface.

Hydroxyapatite crystallization on sodium cholate

Bile acids are known to promote pathological calcification in vivo through the formation of their calcium salts. The role of cholic acid's sodium salt regarding its capability of inducing hydroxyapatite was investigated. It was found that the precipitation of hydroxyapatite was favored by the presence of this organic molecule. In this work, the constant composition kinetics technique was used, which has been proved suitable for the study of the formation of a sparingly soluble salts in the presence of another compound. Kinetic analysis of the nucleation rates as a function of the solution supersaturation yielded a value of 2 for the number of ions forming the critical nucleus. It was suggested that the formation of hydroxyapatite is initiated via interaction of the calcium ions with the negative end of CO bond. The apparent order found from kinetics data was 1.7±0.4 suggesting a surface diffusion-controlled mechanism.

Identification of an NADH-cytochrome b(5) reductase gene from an arachidonic acid-producing fungus, Mortierella alpina 1S-4, by sequencing of the encoding cDNA and heterologous expression in a fungus, Aspergillus oryzae.

Based on the sequence information for bovine and yeast NADH-cytochrome b(5) reductases (CbRs), a DNA fragment was cloned from Mortierella alpina 1S-4 after PCR amplification. This fragment was used as a probe to isolate a cDNA clone with an open reading frame encoding 298 amino acid residues which show marked sequence similarity to CbRs from other sources, such as yeast (Saccharomyces cerevisiae), bovine, human, and rat CbRs. These results suggested that this cDNA is a CbR gene. The results of a structural comparison of the flavin-binding beta-barrel domains of CbRs from various species and that of the M. alpina enzyme suggested that the overall barrel-folding patterns are similar to each other and that a specific arrangement of three highly conserved amino acid residues (i.e., arginine, tyrosine, and serine) plays a role in binding with the flavin (another prosthetic group) through hydrogen bonds. The corresponding genomic gene, which was also cloned from M. alpina 1S-4 by means of a hybridization method with the above probe, had four introns of different sizes. These introns had GT at the 5' end and AG at the 3' end, according to a general GT-AG rule. The expression of the full-length cDNA in a filamentous fungus, Aspergillus oryzae, resulted in an increase (4.7 times) in ferricyanide reduction activity involving the use of NADH as an electron donor in the microsomes. The M. alpina CbR was purified by solubilization of microsomes with cholic acid sodium salt, followed by DEAE-Sephacel, Mono-Q HR 5/5, and AMP-Sepharose 4B affinity column chromatographies; there was a 645-fold increase in the NADH-ferricyanide reductase specific activity. The purified CbR preferred NADH over NADPH as an electron donor. This is the first report of an analysis of this enzyme in filamentous fungi.

Enzymatic degradation of SLN—effect of surfactant and surfactant mixtures

Solid lipid nanoparticles (SLN) show different degradation velocities by the lipolytic enzyme pancreatic lipase as a function of their composition (lipid matrix, stabilizing surfactant). In combination with pancreatic colipase a degradation assay has been developed for studying the degradation behavior. As a measure to follow the degradation the formed free fatty acids have been analyzed using an enzymatic test. In the studies SLN degradation showed dependencies in relation to the length of the fatty acid chains in the triglycerides and the surfactants used for SLN production. The longer the fatty acid chains in the glycerides, the slower the degradation. The influence of surfactants can be degradation accelerating (e.g. cholic acid sodium salt) or a hindering, degradation slowing down effect due to steric stabilization (e.g. Poloxamer 407). As a second steric stabilizer, Tween 80 has been used and the results showed a less pronounced effect on hindering the degradation process than for Poloxamer 407. This result seems to be correlated to the number of ethyleneoxide chains in the molecule. The longer the ethyleneoxide chains are in the molecule, the more hindered is the anchoring of the lipase/colipase complex and consequently the degradation of the SLN. The result can be used to adjust degradation of SLN and consequently drug release in a controlled way.

A synthesis of O -diethylaminoethyl chitosan and its binding ability of cholate and deoxycholate anion in vitro

The aim of this study was to investigate the bile salts adsorption behavior of the diethylaminoethyl (DEAE)-chitosan, in vitro. In order to prepare the DEAE-chitosan, where the DEAE groups are specifically introduced onto 6-OH groups of chitosan with minimal by-product, DEAE-chitin was prepared by reacting chitin with N,N-diethylaminoethyl chloride, and direct N-deacetylation of DEAE-chitin was carried out in 10% of an aqueous sodium hydroxide solution containing sodium borohydride. DEAE chitosan samples were incubated with cholic acid sodium salt and deoxycholic acid sodium salt in a tromethane buffer solution (pH 7.5) for 1 hour, and the bile salts adsorption performances of the samples were examined by HPLC. The modified chitosan samples showed improved bile salts adsorption performances compared to chitosan, and the absorption amount of bile salt to the samples increased with increase in the DEAE groups introduced. In most cases, the adsorption amount of deoxycholate to the samples was greater than that of cholate.