Structural changes in trypsin induced by the bile salts: An effect of amphiphile hydrophobicity.

The present study reports the multi-technique results of the interaction of a series of bile salts, sodium cholate (NaC), sodium taurocholate (NaTC), sodium deoxycholate (NaDC), and sodium taurodeoxycholate (NaTDC) with trypsin under the experimental conditions of 25 °C and pH 7.0. The interactions between trypsin and the bile salts were characterized by the surface tension measurements and various spectroscopic techniques like UV-Visible absorption, steady-state fluorescence, and circular dichroism. The results of surface tension measurements reveal a strong interaction of trypsin (50 µM) with the increasing concentration of bile salts, being higher with the bile salt of greater hydrophobicity. The critical aggregation concentration of bile salts in the presence of trypsin (C1) showed that the bile salts interact strongly with the trypsin in the order of NaTDC > NaDC > NaTC > NaC. UV-visible, steady-state fluorescence, and circular dichroism spectroscopic results confirmed significant unfolding of trypsin due to its interaction with the bile salts, the extent of which followed the same sequence as observed in the surface tension results. It could be concluded that the hydrophobic bile salts that show lower C1 values and have less delocalized charge, are more effective in unfolding the trypsin. The study would help understand the hydrophobicity-driven unfolding of proteins aided by biological surfactants like bile salts and help devise efficient proteolytic enzyme-based detergent formulations and understand the role of such amphiphiles as antimicrobial agents.

Concentration-dependent effects of sodium cholate and deoxycholate bile salts on breast cancer cells proliferation and survival.

Bile acids (BAs) are bioactive molecules that have potential therapeutic interest and their derived salts are used in several pharmaceutical systems. BAs have been associated with tumorigenesis of several tissues including the mammary tissue. Therefore, it is crucial to characterize their effects on cancer cells. The objective of this work was to analyse the molecular and cellular effects of the bile salts sodium cholate and sodium deoxycholate on epithelial breast cancer cell lines. Bile salts (BSs) effects over breast cancer cells viability and proliferation were assessed by MTS and BrdU assays, respectively. Activation of cell signaling mediators was determined by immunobloting. Microscopy was used to analyze cell migration, and cellular and nuclear morphology. Interference of membrane fluidity was studied by generalized polarization and fluorescence anisotropy. BSs preparations were characterized by transmission electron microscopy and dynamic light scattering. Sodium cholate and sodium deoxycholate had dual effects on cell viability, increasing it at the lower concentrations assessed and decreasing it at the highest ones. The increase of cell viability was associated with the promotion of AKT phosphorylation and cyclin D1 expression. High concentrations of bile salts induced apoptosis as well as sustained activation of p38 and AKT. In addition, they affected cell membrane fluidity but not significant effects on cell migration were observed. In conclusion, bile salts have concentration-dependent effects on breast cancer cells, promoting cell proliferation at physiological levels and being cytotoxic at supraphysiological ones. Their effects were associated with the activation of kinases involved in cell signalling.

Coffee parchment as a new dietary fiber ingredient: Functional and physiological characterization.

Coffee parchment was evaluated as a potential dietary fiber ingredient. For this purpose, dietary fiber was extracted by enzymatic and non-enzymatic methods and its physicochemical and in vitro hypoglycemic and hypolipidemic properties were investigated. Results revealed that coffee parchment (flakes and flour) was a good source of insoluble dietary fiber (IDF), mainly composed by xylans (35%), lignin (32%), and cellulose (12%). From results, the IDF extraction seemed not to be required the use of enzymes. Coffee parchment did not stand out by its content of phenolic compounds and antioxidant capacity, but milling process improved them. Due to its physical structure, coffee parchment flakes exhibited high oil holding capacity (3.8 mg L-1), gelation capacity (8%) besides hydration properties, including water holding (3.4 mg L-1), absorption (3.0 mg L-1) and swelling (14 mg L-1) capacities. Its flour and water-insoluble residue showed lower capacities. Nevertheless, these coffee parchment samples presented effective in vitro hypoglycemic properties, showing high glucose adsorption capacity (50-200 mmol L-1), and capacity to decrease its diffusion (13%), and to inhibit α-amylase (52%) that led to lower starch digestibility (until 46%); and also, outstanding in vitro hypolipidemic properties, as inhibition of pancreatic lipase (43%) and binding of cholesterol and sodium cholate (16.6 and 35.3 mg g-1, respectively). These results provide valuable information for the potential use of coffee parchment as new food DF ingredient.

Spectral-fluorescent study of the interaction of polymethine dye probes with biological surfactants - bile salts.

Spectral-fluorescent properties of polymethine dye probes anionic 3,3'-di(sulfopropyl)-4,5,4',5'-dibenzo-9-ethylthiacarbocyanine-betaine (DEC) and cationic 3,3',9-trimethylthiacarbocyanine iodide (Cyan 2) in the presence of biological surfactants, bile salts sodium cholate (NaC), sodium deoxycholate (NaDC) and sodium taurocholate (NaTC), as well as sodium dodecyl sulfate (SDS), have been studied in a wide range of surfactant concentrations. When a surfactant is introduced into a solution of DEC, changes of the spectral-fluorescent properties are observed due to decomposition of dye dimers into cis-monomers and cis-trans conversion of the resulting monomers. In the presence of SDS, both processes occur in parallel, caused by noncovalent interaction of dye monomers with micelles, and mainly occur near the critical micelle concentration (CMC). In contrast, upon the introduction of increasing concentrations of bile salts, decomposition of dye dimers into the monomers begins at lower concentrations than cis-trans conversion. The former process is almost completed at concentrations close to CMC of secondary micelles (CMC2), while the latter process occurs even at concentrations of bile salts much higher than CMC2. Hence, DEC can serve as a probe that permits estimating the value of CMC2 and is indicative of reorganization of secondary micelles upon an increase in bile salt concentration. Aggregation of DEC and Cyan 2 on bile salts is also observed. Since it is observed at relatively low concentrations of bile salts (

Synthetic Biology-Based Solution NMR Studies on Membrane Proteins in Lipid Environments.

Although membrane proteins are in the focus of biochemical research for many decades the general knowledge of this important class is far behind soluble proteins. Despite several recent technical developments, the most challenging feature still is the generation of high-quality samples in environments suitable for the selected application. Reconstitution of membrane proteins into lipid bilayers will generate the most native-like environment and is therefore commonly desired. However, it poses tremendous problems to solution-state NMR analysis due to the dramatic increase in particle size resulting in high rotational correlation times. Nevertheless, a few promising strategies for the solution NMR analysis of membrane inserted proteins are emerging and will be discussed in this chapter. We focus on the generation of membrane protein samples in nanodisc membranes by cell-free systems and will describe the characteristic advantages of that platform in providing tailored protein expression and folding environments. We indicate frequent problems that have to be overcome in cell-free synthesis, nanodisc preparation, and customization for samples dedicated for solution-state NMR. Detailed instructions for sample preparation are given, and solution NMR approaches suitable for membrane proteins in bilayers are compiled. We further discuss the current strategies applied for signal detection from such difficult samples and describe the type of information that can be extracted from the various experiments. In summary, a comprehensive guideline for the analysis of membrane proteins in native-like membrane environments by solution-state NMR techniques will be provided.

Mango phenolics increase the serum apolipoprotein A1/B ratio in rats fed high cholesterol and sodium cholate diets.

BACKGROUND: Serum lipoproteins are in dynamic equilibrium, partially controlled by the apolipoprotein A1 to apolipoprotein B ratio (APOA1/APOB). Freeze-dried mango pulp (FDM) is a rich source of phenolic compounds (MP) and dietary fiber (MF), although their effects on lipoprotein metabolism have not yet been studied. RESULTS: Thirty male Wistar rats were fed with four different isocaloric diets (3.4 kcal g-1 ) for 12 weeks: control diet, high cholesterol (8 g kg-1 ) + sodium cholate (2 g kg-1 ) diet either alone or supplemented with MF (60 g kg-1 ), MP (1 g kg-1 ) or FDM (50 g kg-1 ). MP and FDM reduced food intake, whereas MF and MP tended to increase serum APOA1/APOB ratio, independently of their hepatic gene expression. This suggests that lipoprotein metabolism was favorably altered by mango bioactives, MP also mitigated the non-alcoholic steatohepatitis that resulted from the intake of this diet. CONCLUSION: We propose that phenolics are the most bioactive components of mango pulp, acting as anti-atherogenic and hepatoprotective agents, with a mechanism of action tentatively based on changes to the main protein components of lipoproteins. © 2018 Society of Chemical Industry.

Increasing the Bile Acid Sequestration Performance of Cationic Hydrogels by Using an Advanced/Controlled Polymerization Technique.

PURPOSE: To investigate the influence of the polymerization technique and the content of hydroxyl groups on the performance of new bile acid sequestrants based on PAMPMTA-co-PHEA (PAMPTMA: poly((3-acrylamidopropyl)trimethylammonium chloride); PHEA: poly(2-hydroxyethyl acrylate)) hydrogels. METHODS: PAMPMTA-co-PHEA hydrogels were prepared using either free radical polymerization or supplemental activator and reducing agent atom transfer radical polymerization. The chemical structure and composition of the hydrogels was confirmed by both FTIR and ssNMR. The binding of sodium cholate as the model bile salt was evaluated in simulated intestinal fluid using HPLC. The degradation of the polymers was evaluated in vitro in solutions mimicking the gastrointestinal tract environment. RESULTS: The binding showed that an increase of the amount of HEA in the hydrogel lead to a decrease of the binding capacity. In addition, it was demonstrated for the first time that the hydrogels produced by SARA ATRP presented a higher binding capacity than similar ones produced by FRP. Finally, it was observed that copolymers of PAMPTMA-co-PHEA showed no sign of degradation in solutions mimicking both the stomach and the intestine environment. CONCLUSIONS: The use of an advanced polymerization technique, such as the SARA ATRP, could be beneficial for the preparation of BAS with enhanced performance.

Differential interaction behaviors of an alkaloid drug berberine with various bile salts.

The present study reports a meticulous characterization of the interaction of a potent anti-cancer drug, berberine chloride (BR) with a series of bile salt aggregates having varying hydrophobicity (sodium deoxycholate (NaDC), sodium cholate (NaC), and sodium taurocholate (NaTC)). The absorption spectrum of BR reveals a complex profile comprised of four distinct peaks. Analysis of the modulations of the absorption spectral properties of BR following interaction with the bile salts raising deeper questions unveils a critical insight into the mode of interaction of the cationic drug (BR) with the bile salts; a greater degree of perturbation of the microenvironment of the isoquinolinic part of BR compared to the benzenoid part. The remarkable modulation of the fluorescence profile of BR with added bile salts provides a sensitive indicator for monitoring the interaction scenario. However, an intriguing observation in this context reveals differential fluorescence behavior of BR in various bile salt aggregates, that is, similar observations in NaDC and NaC (which are legitimately interpreted according to the 'two-step association model') in comparison to NaTC. Such contrasting behavior of BR in NaTC aggregates has been rationalized on the basis of the possibility of formation of dye aggregate facilitated because of the proximity of the cationic drug molecules to the anionic headgroup of NaTC bile salt. Surprisingly, our spectroscopic results evidence for binding location of the drug at the interfacial region in all the bile salt aggregates. To this end, the time-resolved fluorescence decay behavior of the drug within various bile salt aggregates has been meticulously studied. The fluorescence decay results are found to be highly sensitive to the structure and size of the bile salt aggregates eventually leading to characterization of the interaction of the drug with the bile salts in excellent corroboration with the steady-state data. Furthermore, the time-resolved fluorescence anisotropy decay measurements yielded insight into the modulation of rotational dynamical behavior of the drug within the bile salt aggregates.

Enhanced Solubility and Bioavailability of Naringenin via Liposomal Nanoformulation: Preparation and In Vitro and In Vivo Evaluations.

This study was aimed at preparing orally administered naringenin-loaded liposome for pharmacokinetic and tissue distribution studies in animal models. The liposomal system, consisting of phospholipid, cholesterol, sodium cholate, and isopropyl myristate, was prepared using the thin-film hydration method. Physicochemical characterization of naringenin-loaded liposome such as particle size, zeta potential, and encapsulation efficiency produced 70.53 ± 1.71 nm, -37.4 ± 7.3 mV, and 72.2 ± 0.8%, respectively. The in vitro release profile of naringenin from the formulation in three different media (HCl solution, pH 1.2; acetate buffer solution, pH 4.5; phosphate buffer solution, pH 6.8) was significantly higher than the free drug. The in vivo studies also revealed an increase in AUC of the naringenin-loaded liposome from 16648.48 to 223754.0 ng·mL-1 h as compared with the free naringenin. Thus, approximately 13.44-fold increase in relative bioavailability was observed in mice after oral administration. The tissue distribution further showed that the formulation was very predominant in the liver. These findings therefore indicated that the liposomal formulation significantly improved the solubility and oral bioavailability of naringenin, thus leading to wider clinical applications.

Investigation of necessity of sodium cholate and minimal required amount of cholesterol for dietary induction of atherosclerosis in microminipigs.

Recently we established a Microminipig (MMPig) model of atherosclerosis induced by high fat/high cholesterol (Cho) diet containing sodium cholate (SC), which is known to cause hepatotoxicity. In the present study, we investigated whether SC is necessary as well as the minimum amount of dietary Cho required to induce atherosclerosis. Experiment A: Six MMPigs were divided into three groups of two, and were fed for 12 weeks as follows: a diet containing 12% fat and 5% Cho with or without 0.7% SC, or the diet including 12% fat and 0.5% Cho with SC. Although each diet induced a similar degree of hypercholesterolemia and atherosclerosis, the liver weights and severity of fatty change in the hepatocytes were maximal in the animals fed 5% Cho and SC. Experiment B: Six MMPigs were divided into two groups of three, and fed for 18 weeks as follows: normal diet, and a diet of increasing dose of Cho (0.03, 0.1, 0.3, 0.5, 1.5 and 5%) for the initial 14 weeks and 0.5% Cho/12% fat diet for the final four weeks. Serum levels of total Cho and low-density lipoprotein-Cho reached a plateau with 0.5% Cho diet, suggesting that the minimum amount of Cho required is 0.5%. The absorption of Cho in MMPigs was enhanced by 0.5% Cho and 12% fat diet compared to the 5% Cho-alone diet. In conclusion, a diet with 0.5% Cho and 12% fat without SC appears to be sufficient to induce atherosclerosis in the MMPig.

Interactions of phenothiazine drugs with bile salts: micellization and binding studies.

An evaluation of the interactions of phenothiazine tranquilizer drugs (promazine hydrochloride; PMZ and promethazine hydrochloride; PMT) with bile salts viz., sodium cholate (NaC) and sodium deoxycholate (NaDC) in aqueous medium, investigated through different physicochemical measurements is presented in this work. The mixed micellization behavior and surface properties of the phenothiazine-bile salt systems have been analyzed by conductivity and surface tension measurements. Application of different theoretical approaches to all the phenothiazine-bile salt mixtures shows a non-ideal behavior. Further, the spectroscopic techniques such as UV-visible and steady state fluorescence have been employed to study the binding of phenothiazines with bile salts. The stoichiometric ratios, binding constants (K), and free energy change (ΔG) for the phenothiazine-bile salt complexes were estimated from the Benesi-Hildebrand (B-H) double reciprocal plots obtained by using the changes in spectral intensities of phenothiazines on addition of bile salts. The results are discussed in the light of use of bile salts as promising drug delivery agents for phenothiazines and hence improve their bioavailabilty.

Improving the absorption of earthworm fibrinolytic enzymes with mucosal enhancers.

Earthworm fibrinolytic enzymes (EFEs), an ideal drug for cardiovascular diseases, have a very low oral bioavailability. In order to improve the absorption of EFEs, six different enhancers were selected to increase the intestinal absorption of EFEs. In vitro (Caco-2 monolayers) and in vivo (mice) experiments were carried out to find the optimum concentration and action time of these enhancers for EFE absorption. We found that EFEs could be transported into blood across intestinal endothelial membrane after administration via intragastric administration with low bioavailability. These results obtained from in vitro experiments were similar to those in vivo. Moreover, menthol and glucose showed absorption enhancement properties with a relatively low cytotoxicity.

A photophysical study on the role of bile salt hydrophobicity in solubilizing amphotericin B aggregates.

Amphotericin B (AmB) is a highly effective antifungal agent and finds utility against a broad spectrum of fungal species. Bile salts are biocompatible biosurfactants, widely used as drug delivery media for many hydrophobic drugs. AmB in the colloidal suspension of sodium deoxycholate (NaDC) is a well-known commercial formulation of AmB. In the present work, the association of AmB with three bile salts, namely sodium cholate, sodium taurodeoxycholate and sodium taurocholate is studied using the photophysical properties of AmB. Selective excitation of monomeric AmB (lambda(ex) 414 nm, lambda(em) 560 nm) and dimeric AmB (lambda(ex) 335 nm, lambda(em) 472 nm) reveal that with increasing concentration of bile salts, the higher aggregates in water disaggregate to form both monomeric and dimeric forms of AmB. This is seen to be a general trend in all the bile salts studied. Results of steady state fluorescence anisotropy and fluorescence lifetimes studies suggest that the interaction between AmB (hydrophobic heptaene face) and bile salts (hydrophobic steroidal face) is essentially hydrophobic.

Effects of various dietary arginine and lysine concentrations on plasma and liver cholesterol concentrations in rats.

BACKGROUND: It has been hypothesized that the arginine:lysine ratio of dietary proteins influences cholesterol concentrations in plasma and liver of men and animals. This study was performed to test this hypothesis in rats by using diets with various concentrations of arginine and lysine, differing in their arginine:lysine ratios. METHODS: Two experiments with growing rats were performed, some of which received diets containing 4.5, 9 or 18 g arginine/kg and 9 or 18 g lysine/kg, respectively, for a period of 21 days. In the first experiment, a cholesterol-free diet was used; in the second experiment, a diet supplemented with cholesterol and sodium cholate as hypercholesterolaemic compounds was used. RESULTS: In experiment 1, increasing the arginine concentration lowered HDL and plasma cholesterol concentration; however, cholesterol concentrations in liver, LDL and VLDL remained unchanged. In experiment 2, increasing the arginine concentration lowered HDL cholesterol and increased liver cholesterol (p<0.05); cholesterol concentrations in plasma, LDL and VLDL remained unchanged. The only effect of the dietary lysine concentration concerned the effect on VLDL and liver cholesterol concentration, which were both lower in rats fed the diets with 18 g lysine/kg than in those fed the diets with 9 g lysine/kg (p<0.05). Varying the dietary arginine:lysine ratio between 0.25 and 2.0 had no influence on cholesterol concentration in LDL and VLDL in both experiments; HDL cholesterol concentration was lowered by increasing this ratio (p<0.05). CONCLUSION: The present study does not support the hypothesis that an increase in the dietary arginine:lysine ratio causes hypocholesterolaemic effects in rats.

Novel O-palmitoylscleroglucan-coated liposomes as drug carriers: development, characterization and interaction with leuprolide.

Polysaccharide-coated liposomes have been studied for their potential use for peptide drug delivery by the oral route because they are able to minimize the disruptive influences on peptide drugs of gastrointestinal fluids. The aim of this work was to synthesize and characterize a modified polysaccharide, O-palmitoylscleroglucan (PSCG), and to coat unilamellar liposomes for oral delivery of peptide drugs. To better evaluate the coating efficiency of PSCG, also scleroglucan (SCG)-coated liposomes were prepared. We studied the surface modification of liposomes and the SCG- and PSCG-coated liposomes were characterized in terms of size, shape, zeta potential, influence of polymer coating on bilayer fluidity, stability in serum, in simulated gastric and intestinal fluids and against sodium cholate and pancreatin. Leuprolide, a synthetic superpotent agonist of luteinizing hormone releasing hormone (LHRH) receptor, was chosen as a model peptide drug. After polymer coating the vesicle dimensions increased and the zeta potential shifted to less negative values. These results indicate that both SCG- and PSCG-coated liposomes surface and DSC results showed that PSCG was anchored on the liposomal surface. The stability of coated-liposomes in SGF, sodium cholate solution and pancreatin solution was increased. From this preliminary in vitro studies, it seems that PSCG-coated liposomes could be considered as a potential carrier for oral administration.

A fluorimetric and circular dichroism study of hemoglobin--effect of pH and anionic amphiphiles.

In this work, bovine hemoglobin (Hb) has been studied mainly by the fluorescence method. pH has been found to exert a profound effect on Hb structure. This has been confirmed by fluorescence and circular dichroism (CD) studies. The pH-induced change in quaternary structure of Hb indirectly affects its secondary structure. This in turn affects ligand binding to Hb at various pH. The binding of two amphiphiles, a bile salt and a surfactant, have been investigated. The pH-induced structural modification of Hb has been confirmed by studies with the well-known denaturant urea and the polarity probe ANS, which has been used as an extrinsic fluorophore.

In vitro and in vivo transfection efficiency of a novel ultradeformable cationic liposome.

Cationic lipids have been often used as one of the major components in making most promising non-viral gene delivery systems, whereas sodium cholate, a surfactant so-called edge activator has been used in preparing ultradeformable and ultraflexible liposomes called Transfersomes. Using both a cationic lipid, DOTAP and sodium cholate, a novel formulation of ultradeformable cationic liposome (UCL) has been prepared. The average particle size of this formulation was approximately 80 nm. The physical and chemical stabilities at two different temperatures (4 degrees C and 20 degrees C) were also evaluated for 60 days. The ultradeformability of new formulation was also assessed, and it has been proved that the formulation is deformable. In vitro transfection efficiency of plasmid DNA/UCL was assessed by the expression of green fluorescent protein (GFP) in four cell lines, OVCAR-3 (human ovarian carcinoma cells), HepG2 (human hepatoma cells), H-1299 (human lung carcinoma cells) and T98G (human brain carcinoma cells). The optimal ratio of DNA to liposome for maximal transfection efficiency was 1:14 (w/w) in all the cell lines except for the human brain carcinoma cells. The same formulation was tested for in vivo transfection efficiency and its retention time within the organs by applying the DNA/UCL complexes on hair-removed dorsal skin of mice non-invasively. It was found that genes were transported into several organs for 6 days once applied on intact skin.

Ligand association with the rabbit kidney and brain Y1, Y2 and Y5-like neuropeptide Y (NPY) receptors shows large subtype-related differences in sensitivity to chaotropic and alkylating agents.

The binding to rabbit kidney or hypothalamic particulates of the subtype-selective neuropeptide Y (NPY) receptor ligands [125I](Leu31,Pro34)hPYY (as Y1 site label at 2 nM human pancreatic polypeptide (hPP)), [125I]-hPYY(3-36) (Y2 label), and [125I]-hPP (Y5 label) displayed great differences in sensitivity to alkylators and chaotropic agents. Sensitivity to a nonionic chaotrope, urea, was much higher for the Y1 binding than for the Y5-like binding or the Y2 binding. The non-selective alkylator N-ethylmaleimide (NEM) and several alkylators selective for aminergic receptors were much more efficacious against the Y1 relative to the Y2 binding. Similar differences could be confirmed with the attachment of Y1 and Y2-selective tracers to CHO cells expressing the cloned guinea-pig Y1 or Y2 receptors. The Y5-like binding was quite insensitive to NEM, but sensitive to chloroethylclonidine (CEC) and prazobind, which were less potent at the Y1, and especially at the Y2 site. The unrestricted-access alkylator 2-aminoethyl methanethiosulfonate inhibited the binding to all subtypes, while the restricted-access agent 2-(trimethylammonium)ethylmethanethiosulfonate poorly inhibited the Y5-like binding, or the guanine nucleotide-insensitive Y2 binding. These results are compatible with an active conformation of the Y5-like site dependent on maintenance of a shared hydrophobic cavity. The Y2 sites resistant to guanosine polyphosphates and restricted-access alkylators were detected mainly in particulates slowly solubilized by cholate at 0-5 degrees C; these sites could be clustered.