NMR Study of Water-Soluble Carotenoid Crocin: Formation of Mixed Micelles, Interaction with Lipid Membrane and Antioxidant Activity.

Crocin is a unique water-soluble carotenoid found in crocus and gardenia flowers. Crocin has been shown to have a variety of pharmacological activities, such as antioxidant, anti-cancer, memory improvement, antidepressant, anti-ischemia, blood pressure lowering and aphrodisiac, gene protection and detoxification activities. Due to their amphiphilicity, crocin molecules form concentration-dependent self-associates (micelles) in a water solution. In the present study, using various NMR techniques (T2 relaxation and selective gradient NOESY), we have demonstrated that crocin forms mixed micelles with water-soluble drug delivery system glycyrrhizin and linoleic acid molecules. Note, that the spin-spin T2 relaxation time and NOESY spectroscopy are very sensitive to intermolecular interactions and molecular diffusion mobility. The second purpose of this work was the elucidation of the interaction of crocin with a model lipid membrane using NMR techniques and a molecular dynamics simulation and its effects on lipid oxidation. It was shown that the crocin molecule is located near the surface of the lipid bilayer and effectively protects lipids from oxidation by peroxyl radicals. The role of glycyrrhizin and vitamin C in metal-induced lipid oxidation was also elucidated. The results of this study may be useful for expanding the field of application of crocin in medicine and in the food industry.

Increased Free Radical Generation during the Interaction of a Quinone-Quinoline Chelator with Metal Ions and the Enhancing Effect of Light.

Schiff bases and similar molecules forming metal complexes may cause redox effects, which may also be influenced by light. Anthraquinones such as doxorubicin and idarubicin are widely used antitumor agents, which can generate reactive oxygen species (ROS), stimulated by both the presence of iron and copper ions and also by light. The generated ROS can cause DNA scission, cell membrane oxidation, and many other toxic effects. The redox activity of the quinone-quinoline chelator 2-phenyl-4-(butylamino)naphtho [2,3-h]quinoline-7,12-dione (Q1) was investigated in the presence of iron, copper, and zinc. The influence of light in these interactions was also examined. The chemically induced dynamic nuclear polarization (CIDNP), nuclear magnetic resonance (NMR), and electron paramagnetic resonance (EPR) methods were used to elucidate the molecular changes and ROS generation effects of the Q1 metal interactions. A model electron transfer reaction system between 1,4-dihydropyridine and Q1 was utilized to demonstrate that the chelate complexes of Q1 with both Fe(III) and Cu(II) ions were more redox active than Q1 itself. Similarly, CIDNP and NMR data showed that the concentration dependence of the free radicals yield is much higher in the presence of Fe(III) and Cu(II) ions, in comparison to Zn(II), and also that it increased in the presence of light. These findings underline the role of transition metal ions and Q1 in cyclic redox chain reactions and increase the prospect of the development of copper- and iron-based chelating agents, including Q1 and its derivatives, for anticancer therapy. Furthermore, these findings also signify the effect of light on enhancing ROS formation by Q1 and the prospect of utilizing such information for designing target specific anticancer drugs for photodynamic therapy.

Localization of the ibuprofen molecule in model lipid membranes revealed by spin-label-enhanced NMR relaxation.

Non-steroidal anti-inflammatory drugs (NSAIDs) have antipyretic, anti-inflammatory and analgesic effects, and can be used in the treatment of various diseases. These drugs have also a number of side effects, which may be related to their interaction with lipid membranes. In this study, we use the spin-labeled NSAID ibuprofen (ibuprofen-SL) as a relaxation enhancer to study its interaction with model lipid membranes employing liquid-state 1H NMR at 500 MHz. The high magnetic moment of unpaired electron in the spin label made it possible to reduce the concentration of the studied drug in the membrane to tenths of a mole percent. As model membranes, unilamellar POPC liposomes and bicelles consisting of a 2:1 mixture of DHPC:DMPC or DHPC:POPC lipids were used. An increase in the rate of proton spin-lattice relaxation, T1-1, selectively detected for protons at different positions in the lipid molecule, showed that ibuprofen-SL is localized in the hydrophobic part of the lipid bilayer. As the concentration of ibuprofen-SL increases to 0.5 mol%, the distribution of positions of ibuprofen-SL across the bilayer becomes wider. In the presence of 20 mol% of cholesterol, ibuprofen-SL is displaced from the core of the membrane to a region closer to the head group of the bilayer. This displacement was also confirmed by the NMR NOESY experiment conducted with unlabeled ibuprofen. For bilayers containing unsaturated POPC lipids, the distribution of ibuprofen positions across the bilayer becomes narrower compared to the presence of saturated DMPC lipids.

The Interaction of the Transmembrane Domain of SARS-CoV-2 E-Protein with Glycyrrhizic Acid in Lipid Bilayer.

The interaction of the transmembrane domain of SARS-CoV-2 E-protein with glycyrrhizic acid in a model lipid bilayer (small isotropic bicelles) is demonstrated using various NMR techniques. Glycyrrhizic acid (GA) is the main active component of licorice root, and it shows antiviral activity against various enveloped viruses, including coronavirus. It is suggested that GA can influence the stage of fusion between the viral particle and the host cell by incorporating into the membrane. Using NMR spectroscopy, it was shown that the GA molecule penetrates into the lipid bilayer in a protonated state, but localizes on the bilayer surface in a deprotonated state. The transmembrane domain of SARS-CoV-2 E-protein facilitates deeper GA penetration into the hydrophobic region of bicelles at both acidic and neutral pH and promotes the self-association of GA at neutral pH. Phenylalanine residues of the E-protein interact with GA molecules inside the lipid bilayer at neutral pH. Furthermore, GA influences the mobility of the transmembrane domain of SARS-CoV-2 E-protein in the bilayer. These data provide deeper insight into the molecular mechanism of antiviral activity of glycyrrhizic acid.

Metal Complexes of Omadine (N-Hydroxypyridine-2-thione): Differences of Antioxidant and Pro-Oxidant Behavior in Light and Dark Conditions with Possible Toxicity Implications.

Omadine or N-hydroxypyridine-2-thione and its metal complexes are widely used in medicine and show bactericidal, fungicidal, anticancer, and photochemical activity. The redox activity of omadine complexes with iron, copper, and zinc on lipid peroxidation under light and dark conditions has been investigated. The monitoring of the oxidation of linoleic acid micelles, resembling a model of lipid membrane, was carried out using nuclear magnetic resonance (1H-NMR). It has been shown that the omadine-zinc complex can induce the oxidation of linoleic acid under light irradiation, whereas the complexes with iron and copper are photochemically stable. All the chelating complexes of omadine appear to be redox-inactive in the presence of hydrogen peroxide under dark conditions. These findings suggest that omadine can demonstrate antioxidant behavior in processes involving reactive oxygen species generation induced by transition metals (Fenton and photo-Fenton reactions). However, the omadine complex with zinc, which is widely used in shampoos and ointments, is photochemically active and may cause oxidative cell membrane damage when exposed to light, with possible implications to health.

The Interaction of Anthracycline Based Quinone-Chelators with Model Lipid Membranes: 1H NMR and MD Study.

Anthracycline antibiotics, e.g., doxorubicin, daunomycin, and other anthraquinones, are an important family of antitumor agents widely used in chemotherapy, which is currently the principal method for treating many malignancies. Thus, development of improved antitumor drugs with enhanced efficacy remains a high priority. Interaction of anthraquinone-based anticancer drugs with cell membranes attracts significant attention due to its importance in the eventual overcoming of multidrug resistance (MDR). The use of drugs able to accumulate in the cell membrane is one of the possible ways of overcoming MDR. In the present work, the aspects of interaction of anthraquinone 2-phenyl-4-(butylamino)naphtho[2,3-h]quinoline-7,12-dione) (Q1) with a model membrane were studied by means of NMR and molecular dynamics simulations. A fundamental shortcoming of anthracycline antibiotics is their high cardiotoxicity caused by reactive oxygen species (ROS). The important feature of Q1 is its ability to chelate transition metal ions responsible for ROS generation in vivo. In the present study, we have shown that Q1 and its chelating complexes penetrated into the lipid membrane and were located in the hydrophobic part of the bilayer near the bilayer surface. The chelate complex formation of Q1 with metal ions increased its penetration ability. In addition, it was found that the interaction of Q1 with lipid molecules could influence lipid mobility in the bilayer. The obtained results have an impact on the understanding of molecular mechanisms of Q1 biological activity.

Holmium Complex with Phospholipids as 1H NMR Relaxational Sensor of Temperature and Viscosity.

The sensitivity of Ho-phospholipid complexes to changes in the membrane viscosity of liposomes was checked. An increase in viscosity was observed for DPPC and DMPC near the phase-transition temperature. Ho-phospholipid complexes could be used as sensors of local membrane viscosity in NMR and MRI technologies.

Stereoselectivity of Interaction of Nonsteroidal Anti-Inflammatory Drug S-Ketoprofen with L/D-Tryptophan in Phospholipid Membranes.

The mechanisms of stereoselectivity of the interaction of chiral drugs with active sites of enzymes and cell receptors attract significant attention. The first reason is the difference in therapeutic activity of the enantiomers of the common drugs. Another reason is the interest in the role of chiral inversion of amino acids involved in various peptides in the development of many diseases including Alzheimer's, Parkinson's, type II diabetes, and a number of other pathological conditions. In our study we use elementary chemical process-electron transfer (ET) to simulate individual stages of ligand-receptor and enzyme-substrate interactions. In particular, previous studies of photoinduced ET in chiral donor-acceptor dyads consisting of the nonsteroidal anti-inflammatory drug (R/S)-ketoprofen and (L)-tryptophan show the stereo and spin selectivity of ET in diastereomers. The present study is devoted to the interaction of (S)-ketoprofen with L- and D-enantiomers of tryptophan in homogeneous aqueous solution and in phospholipid membranes. The study was done using the NMR technique and molecular modeling. These approaches confirm efficient penetration of ketoprofen into the lipid bilayer and binding with tryptophan molecule. The short-lived paramagnetic intermediates formed during the photoinduced ET from electron donor tryptophan to ketoprofen have been detected using the chemically induced dynamic nuclear polarization (CIDNP) technique. It was found that S-ketoprofen interacts stereoselectively with tryptophan enantiomers in the lipid membrane. The formation of the ketyl radical of ketoprofen under irradiation leads to the oxidation of membrane lipids and may be the cause of ketoprofen phototoxicity. However, in contrast to a homogeneous solution in phosphate buffer saline, where the amino acid tryptophan accelerates the photodecomposition of KP due to intramolecular hydrogen transfer, tryptophan in a lipid membrane significantly reduces the rate of photodegradation due to a reversible electron (or hydrogen) transfer reaction. The stereoselectivity in the rate of KP and lipids decomposition under UV irradiation of S-ketoprofen in the presence of tryptophan enantiomers in lipid bilayer has been detected.

Photoinduced Oxidation of Lipid Membranes in the Presence of the Nonsteroidal Anti-Inflammatory Drug Ketoprofen.

The damage of cell membranes induced by photosensitive drugs has attracted the significant attention of researchers in various fields of medicine. Ketoprofen (KP) is known to be the most photosensitive among the nonsteroidal anti-inflammatory drugs. The phototoxic side effects of KP and other non-steroidal anti-inflammatory drugs are associated with the action of free radicals, but there is insufficient information about the nature of these radicals. In the present study, free radicals formed upon KP irradiation within lipid membranes were studied using nuclear magnetic resonance (NMR) and chemically induced dynamic nuclear polarization (CIDNP) methods, as well as a molecular dynamics simulation. Our study confirmed the effective penetration of KP into the lipid bilayer and showed a significant effect of the nature of the medium on the photolysis mechanism. While, in a homogeneous solution, the main channel of KP photolysis is free radical-mediated monomolecular decomposition with formation of radical pairs of benzyl and CO2H● radicals, then, in the lipid membrane, the reaction route shifts towards the bimolecular reaction of KP photoreduction. In addition, the effect of the presence an electron donor (the amino acid tryptophan) on lipid oxidation has been studied. It was found that photoreaction of KP with tryptophan proceeds more efficiently than with lipid molecules.

Mechanistic Insights of Chelator Complexes with Essential Transition Metals: Antioxidant/Pro-Oxidant Activity and Applications in Medicine.

The antioxidant/pro-oxidant activity of drugs and dietary molecules and their role in the maintenance of redox homeostasis, as well as the implications in health and different diseases, have not yet been fully evaluated. In particular, the redox activity and other interactions of drugs with essential redox metal ions, such as iron and copper, need further investigation. These metal ions are ubiquitous in human nutrition but also widely found in dietary supplements and appear to exert major effects on redox homeostasis in health, but also on many diseases of free radical pathology. In this context, the redox mechanistic insights of mainly three prototype groups of drugs, namely alpha-ketohydroxypyridines (alpha-hydroxypyridones), e.g., deferiprone, anthraquinones, e.g., doxorubicin and thiosemicarbazones, e.g., triapine and their metal complexes were examined; details of the mechanisms of their redox activity were reviewed, with emphasis on the biological implications and potential clinical applications, including anticancer activity. Furthermore, the redox properties of these three classes of chelators were compared to those of the iron chelating drugs and also to vitamin C, with an emphasis on their potential clinical interactions and future clinical application prospects in cancer, neurodegenerative and other diseases.

The Interplay of Ascorbic Acid with Quinones-Chelators-Influence on Lipid Peroxidation: Insight into Anticancer Activity.

Ascorbic acid is a multifaceted compound that can perform both antioxidant and pro-oxidant activities in the redox reactions induced by transition metal ions, so its role in nature and especially in the human body is still the subject of debate. In the present study, we have examined the influence of ascorbic acid on lipid peroxidation in a model system that mimics the cell membrane, namely micelles of linoleic acid (LA), induced by chelate complexes of iron and copper ions with quinone-chelator 2-phenyl-4-(butylamino)-naphtholquinoline-7,12-dione (Q1). This quinone effectively generates reactive oxygen species and semiquinone radicals inside cancer cells via a cycling redox reaction. Here it was demonstrated that in the absence of quinone-chelator ascorbic acid significantly accelerates the lipid peroxidation induced by both Fe(II) and Cu(II) ions. It has been shown also that Q1 chelate complexes with Fe(II) and Cu(II) ions are redox active in the LA micelles oxidation. No effect of ascorbate was detected on the reactivity of chelate complex with Fe(II) ions. On the other hand, ascorbate performs pro-oxidant activity in Q1-Cu(II) complex induced reaction. We can conclude that ascorbate-driven redox cycling of Q1 may promote its anti-tumor activity.

Antioxidant Activity of Deferasirox and Its Metal Complexes in Model Systems of Oxidative Damage: Comparison with Deferiprone.

Deferasirox is an orally active, lipophilic iron chelating drug used on thousands of patients worldwide for the treatment of transfusional iron overload. The essential transition metals iron and copper are the primary catalysts of reactive oxygen species and oxidative damage in biological systems. The redox effects of deferasirox and its metal complexes with iron, copper and other metals are of pharmacological, toxicological, biological and physiological importance. Several molecular model systems of oxidative damage caused by iron and copper catalysis including the oxidation of ascorbic acid, the peroxidation of linoleic acid micelles and the oxidation of dihydropyridine have been investigated in the presence of deferasirox using UV-visible and NMR spectroscopy. Deferasirox has shown antioxidant activity in all three model systems, causing substantial reduction in the rate of oxidation and oxidative damage. Deferasirox showed the greatest antioxidant activity in the oxidation of ascorbic acid with the participation of iron ions and reduced the reaction rate by about a 100 times. Overall, deferasirox appears to have lower affinity for copper in comparison to iron. Comparative studies of the antioxidant activity of deferasirox and the hydrophilic oral iron chelating drug deferiprone in the peroxidation of linoleic acid micelles showed lower efficiency of deferasirox in comparison to deferiprone.

pH-Sensitive Glycyrrhizin Based Vesicles for Nifedipine Delivery.

Glycyrrhizic acid, or glycyrrhizin (GA), a major active component of licorice root, has been widely used in traditional Chinese and Japanese medicine since ancient times. However, only in the last decades has a novel and unusual property of the GA been discovered to form water-soluble, supramolecular complexes with a variety of lipophilic drugs. These complexes show significant advantages over other known delivery systems, in particular, due to strong pH sensitivity, the properties of GA self-associates. In the present study, a supramolecular complex formation of the hypotensive and antiarrhythmic drug nifedipine with GA has been studied at different pH values, corresponding to the different degrees of GA dissociation, including a fully dissociated state of GA. Both NMR experiments and molecular dynamics simulations demonstrate the existence of the nifedipine complex with GA at all dissociation states of GA. However, optical absorption experiments show the decrease of complex stability and solubility at pH > 6 when the GA molecule is fully deprotonated. It means the higher release rate of the drug in a neutral and basic environment compared with acid media. These results could form the basis of follow-up studies of GA self-associates as pH-controlled drug delivery systems.

Optical Configuration Effect on the Structure and Reactivity of Diastereomers Revealed by Spin Effects and Molecular Dynamics Calculations.

The peculiarities of spin effects in photoinduced electron transfer (ET) in diastereomers of donor-acceptor dyads are considered in order to study the influence of chirality on reactivity. Thus, the spin selectivity-the difference between the enhancement coefficients of chemically induced dynamic nuclear polarization (CIDNP)-of the dyad's diastereomers reflects the difference in the spin density distribution in its paramagnetic precursors that appears upon UV irradiation. In addition, the CIDNP coefficient itself has demonstrated a high sensitivity to the change of chiral centers: when one center is changed, the hyperpolarization of all polarized nuclei of the molecule is affected. The article analyzes the experimental values of spin selectivity based on CIDNP calculations and molecular dynamic modeling data in order to reveal the effect of optical configuration on the structure and reactivity of diastereomers. In this way, we succeeded in tracing the differences in dyads with L- and D-tryptophan as an electron donor. Since the replacement of L-amino acid with D-analog in specific proteins is believed to be the cause of Alzheimer's and Parkinson's diseases, spin effects and molecular dynamic simulation in model dyads can be a useful tool for investigating the nature of this phenomenon.

Effect of Glycyrrhizic Acid and Arabinogalactan on the Membrane Potential of Rat Thymocytes Studied by Potential-Sensitive Fluorescent Probe.

The effect of the natural saponin glycyrrhizic acid (GA) and polysaccharide arabinogalactan (AG) on the transmembrane potential of rat thymocytes was investigated using the potential-sensitive fluorescent probe 4-(p-dimethylaminostyryl)-1-methylpyridinium (DSM). Incubation of cells with GA in micellar form resulted in a decrease of the amplitude of observed fluorescence kinetics that points out to a decrease of the transmembrane potential. The proposed mechanism is an increase of membrane ion permeability (passive ion transport) of the plasma cell membrane due to GA incorporation. The incorporation of GA molecules into the cell membrane is extremely sensitive to the degree of GA dissociation. The neutral form of glycyrrhizic acid enters the lipid bilayer in contrast to the deprotonated anionic form. The incubation of rat thymocytes with anionic form of GA, namely with its disodium salt, has no effect on the fluorescence kinetics. The possible reasons of this phenomenon are discussed in the light of the nuclear magnetic resonance (NMR) and molecular dynamics (MD) data. The treatment of thymocytes with AG affects only the initial rate of the probe incorporation. The proposed mechanism is that AG covers the surface of the cell membrane and forms a barrier for the probe. Additionally, our experiments demonstrated that both polysaccharide AG and GA in the neutral form (but not Na2GA) effectively capture the cationic probe in an aqueous solution and then deliver it to the cell membrane.

Arabinogalactan and glycyrrhizin based nanopesticides as novel delivery systems for plant protection.

During the past decade, nanotechnologies opened a new era in delivery of plant protection products through the development of nanosized controlled release systems, such as polymer nanoparticles, micelles, and so on using a wide variety of materials. To increase the pesticides penetration into the grain under the presowing seed treatment, a new approach based on non-covalent associate preparation with natural polysaccharides and oligosaccharides as delivery systems (DSs) was applied. Earlier, this approach was tested on antidote 1,8-naphthalic anhydride (NA). Enhancement of the NA solubility and penetration into the barley and wheat seeds had been demonstrated. In the present study, these DSs were used to prepare nanocomposites of pesticides (tebuconazole, imidacloprid, imazalil, prochloraz). The composite formation of the pesticides with poly- and oligosaccharides was proved by NMR relaxation method. Enhancement of the pesticides solubility and improvement of its penetration into the seeds of corn and rapeseeds has been detected. The strongest enhancement of penetration ability was observed for arabinogalactan nanocomposites: 5-folds for tebuconazole and imidacloprid, and more than 10-folds for imazalil and prochloraz. Our data show that the effect of polysaccharides and oligosaccharides on the nanopesticide penetration might be associated with the solubility enhancement, affinity of DSs to the surface of grains, and the modification of cell membranes by poly- and oligosaccharides.

Glycyrrhizin-Assisted Transport of Praziquantel Anthelmintic Drug through the Lipid Membrane: An Experiment and MD Simulation.

Praziquantel (PZQ) is one of the most widespread anthelmintic drugs. However, the frequent insufficient application of PZQ after oral administration is associated with its low solubility, penetration rate, and bioavailability. In the present study, the permeation of PZQ through a 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC) membrane was investigated to probe glycyrrhizin-assisted transport. Glycyrrhizin (or glycyrrhizic acid, GA), a natural saponin, shows the ability to enhance the therapeutic activity of various drugs when it is used as a drug delivery system. However, the molecular mechanism of this effect is still under debate. In the present study, the transport rate was measured experimentally by a parallel artificial membrane permeation assay (PAMPA) and molecular dynamics (MD) simulation with DOPC lipid bilayers. The formation of the noncovalent supramolecular complex of PZQ with disodium salt of GA (Na2GA) in an aqueous solution was proved by the NMR relaxation technique. PAMPA experiments show a strong increase in the amount of the penetrating praziquantel molecules in comparison with a saturated aqueous solution of pure drug used as a control. MD simulation of PZQ penetration through the bilayer demonstrates an increase in permeability into the membrane in the presence of a glycyrrhizin molecule. A decrease in the free energy barrier in the middle of the lipid bilayer was obtained, associated with the hydrogen bond between PZQ and GA. Also, GA reduces the local bilayer surface resistance to penetration of PZQ by rearranging the surface lipid headgroups. This study clarifies the mechanism of increasing the drug's bioavailability in the presence of glycyrrhizin.

Solubilization and stabilization of macular carotenoids by water soluble oligosaccharides and polysaccharides.

Xanthophyll carotenoids zeaxanthin and lutein play a special role in the prevention and treatment of visual diseases. These carotenoids are not produced by the human body and must be consumed in the diet. On the other hand, extremely low water solubility of these carotenoids and their instability restrict their practical application as components of food or medicinal formulations. Preparation of supramolecular complexes of zeaxanthin and lutein with glycyrrhizic acid, its disodium salt and the natural polysaccharide arabinogalactan allows one to minimize the aforementioned disadvantages when carotenoids are used in food processing as well as for production of therapeutic formulations with enhanced solubility and stability. In the present study, the formation of supramolecular complexes was investigated by NMR relaxation, surface plasmon resonance (SPR) and optical absorption techniques. The complexes increase carotenoid solubility more than 1000-fold. The kinetics of carotenoid decay in reactions with ozone molecules, hydroperoxyl radicals and metal ions were measured in water and organic solutions, and significant increases in oxidation stability of lutein and zeaxanthin in arabinogalactan and glycyrrhizin complexes were detected.