Ascorbate-and iron-driven redox activity of Dp44mT and Emodin facilitates peroxidation of micelles and bicelles.

BACKGROUND: Iron (Fe)-induced oxidative stress leads to reactive oxygen species that damage biomembranes, with this mechanism being involved in the activity of some anti-cancer chemotherapeutics. METHODS: Herein, we compared the effect of the ligand, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), or the potential ligand, Emodin, on Fe-catalyzed lipid peroxidation in cell membrane models (micelles and bicelles). These studies were performed in the presence of hydrogen peroxide (H2O2) and the absence or presence of ascorbate. RESULTS: In the absence of ascorbate, Fe(II)/Emodin mixtures incubated with H2O2 demonstrated slight pro-oxidant properties on micelles versus Fe(II) alone, while the Fe(III)-Dp44mT complex exhibited marked antioxidant properties. Examining more physiologically relevant phospholipid-containing bicelles, the Fe(II)- and Fe(III)-Dp44mT complexes demonstrated antioxidant activity without ascorbate. Upon adding ascorbate, there was a significant increase in the peroxidation of micelles and bicelles in the presence of unchelated Fe(II) and H2O2. The addition of ascorbate to Fe(III)-Dp44mT substantially increased the peroxidation of micelles and bicelles, with the Fe(III)-Dp44mT complex being reduced by ascorbate to the Fe(II) state, explaining the increased reactivity. Electron paramagnetic resonance spectroscopy demonstrated ascorbyl radical anion generation after mixing ascorbate and Emodin, with signal intensity being enhanced by H2O2. This finding suggested Emodin semiquinone radical formation that could play a role in its reactivity via ascorbate-driven redox cycling. Examining cultured melanoma cells in vitro, ascorbate at pharmacological levels enhanced the anti-proliferative activity of Dp44mT and Emodin. CONCLUSIONS AND GENERAL SIGNIFICANCE: Ascorbate-driven redox cycling of Dp44mT and Emodin promotes their anti-proliferative activity.

Mechanism of the enhancing effect of glycyrrhizin on nifedipine penetration through a lipid membrane.

The saponin glycyrrhizin from liquorice root shows the ability to enhance the therapeutic activity of other drugs when used as a drug delivery system. Due to its amphiphilic properties, glycyrrhizin can form self-associates (dimers, micelles) and supramolecular complexes with a wide range of hydrophobic drugs, which leads to an increase in their solubility, stability and bioavailability. That is why the mechanism of the biological activity of glycyrrhizin is of considerable interest and has been the subject of intensive physical and chemical research in the last decade. Two mechanisms have been proposed to explain the effect of glycyrrhizin on drug bioavailability, namely, the increase in drug solubility in water and enhancement of the membrane permeability. Interest in the membrane-modifying ability of glycyrrhizic acid (GA) is also growing at present due to its recently discovered antiviral activity against SARS-CoV-2 Bailly and Vergoten (2020) [1]. In the present study, the passive permeability of the DOPC lipid membrane for the calcium channel blocker nifedipine was elucidated by parallel artificial membrane permeability assay (PAMPA) and full atomistic molecular dynamics (MD) simulation with free energy calculations. PAMPA experiments show a remarkable increase in the amount of nifedipine (NF) permeated with glycyrrhizin compared to free NF. In previous studies, we have shown using MD techniques that glycyrrhizin molecules can integrate into the lipid bilayer. In this study, MD simulation demonstrates a significant decrease in the energy barrier of NF penetration through the lipid bilayer in the presence of glycyrrhizin both in the pure DOPC membrane and in the membrane with cholesterol. This effect can be explained by the formation of hydrogen bonds between NF and GA in the middle of the bilayer.

Effect of glycyrrhizic acid on phospholipid membranes in media with different pH.

Glycyrrhizic acid (GA) is the active ingredient in licorice root, which exhibits a wide range of biological activities, including anti-inflammatory and antiviral activities. In particular, the virus-inhibiting effect of GA on SARS-associated coronavirus was demonstrated. In addition, GA was found to be capable of increasing bioaccessibility of other drugs when used together. All these effects can be based on the ability of GA to incorporate into cell membranes and change their physical and functional properties. One of the possible mechanisms of the antiviral action of GA against COVID-19 is also considered to be the prevention of fusion of the virus envelope with the plasma membrane of the host cell. The interaction of GA with model lipid membranes was studied by the NMR method. Different factors influencing the incorporation of the GA molecule into the lipid bilayer (phospholipid structure, pH of the medium) were examined.

Glycyrrhizin-induced changes in phospholipid dynamics studied by 1H NMR and MD simulation.

Phospholipid bilayer constitutes the basis of the cell membrane. Any changes in its structure and dynamics could significantly affect the properties and functions of the cell membrane and associated proteins. It could, in its turn, affect the mechanism and strength of drug-membrane interaction. Phase transitions in lipid bilayer play an important role in cell life and in transmembrane transport of ions and drug molecules. In the present study we have tried to clarify the mechanism of glycyrrhizin bioactivity by the study of its influence on the lipid dynamics and phase transition of the lipid bilayer. For this purpose, a combination of nuclear magnetic resonance (NMR) and molecular dynamic (MD) simulations was used. Glycyrrhizin is the saponin extracted from licorice root. It displays a wide spectrum of biological activity and is frequently used in traditional medicine since ancient times. Now glycyrrhizin attracts additional attention as a novel multifunctional drug delivery system. We have established that glycyrrhizin interaction with dipalmitoylphosphatidylcholine lipid bilayers leads to changes in lipid mobility and phase transition temperature. NMR and MD results demonstrated that a glycyrrhizin molecule is able to integrate into a lipid bilayer and form stable aggregates inside. We hypothesize that surface curvatures caused by local changes in the lipid composition and the presence of phase boundaries might affect the permeability of the cell membrane.

Effective inhibition of copper-catalyzed production of hydroxyl radicals by deferiprone.

Copper ions can catalyze the production of free oxygen radicals (•OH and •OOH) similar to iron ions. The capacity to initiate oxidative damage is most commonly attributed to Cu-induced toxicity in copper-related diseases where there is an increase in copper levels and also when Cu homeostasis and regulation are disrupted. An antioxidant/chelator inhibiting Cu-induced oxidative damage could play a significant role in the treatment of such Cu-related diseases. Deferiprone has high affinity for copper binding and can be considered for the potential treatment of copper toxicity and overloading conditions, such as Wilson's disease. In the present study, the ability of deferiprone to inhibit the production of hydroxyl radicals catalyzed by copper ions was elucidated using an Electron Paramagnetic Resonance (EPR) spin trapping technique. The values of g-factors and hyperfine splitting constants were calculated for Cu(II)-deferiprone 1:1 complex: (a = 58.5 G, g = 2.1667) and 1:2 complex: (a = 73.0 G, g = 2.1378). The TMIO spin trap (2,2,4-trimethyl-2H-imidazole-1-oxide) was used for the detection of free radicals formed in Fenton-like copper-catalyzed reactions. It was demonstrated that the interaction of deferiprone with Cu2+ ions completely inhibited hydroxyl radical (•OH) production in the presence of hydrogen peroxide. It was found also that deferiprone inhibits Cu-induced oxidation of linoleic acid in micellar solution. In addition to existing data for water solutions, the affinity of deferiprone for copper binding in non-aqueous environment has been elucidated.

Glycyrrhizic acid as a multifunctional drug carrier - From physicochemical properties to biomedical applications: A modern insight on the ancient drug.

Glycyrrhizic acid is the main active component of Licorice root which has been known in traditional Chinese and Japanese medicine since ancient times. In these cultures glycyrrhizic acid (GA) is one of the most frequently used drugs. However, only in 21-st century a novel unusual property of the GA to enhance the activity of other drugs has been discovered. The review describes briefly the experimental evidences of wide spectrum of own biological activities of glycyrrhizic acid as well as discusses the possible mechanisms of the ability of GA to enhance the activity of other drugs. We have shown that due to its amphiphilic nature GA is able to form self-associates in aqueous and non-aqueous media, as well as water soluble complexes with a wide range of lipophilic drugs. The main purpose of our review is to focus reader's attention on physicochemical studies of the molecular mechanisms of GA activity as a drug delivery system (DDS). In our opinion, the most intriguing feature of glycyrrhizic acid which might be the key factor in its therapeutic activity is the ability of GA to incorporate into the lipid bilayer and to increase the membrane fluidity and permeability. The ability of biomolecules and their aggregates to change the properties of cell membranes is of great significance, from both fundamental and practical points of view.

Physicochemical Properties and Anti-Opisthorchosis Effect of Mechanochemically Synthesized Solid Compositions of Praziquantel with Glycyrrhizic Acid Disodium Salt.

The mechanochemical preparation of solid compositions of praziquantel with plant saponin (glycyrrhizic acid disodium salt) is described. The study of a number of physicochemical parameters showed that dissolving solid compositions in water is accompanied by the inclusion of praziquantel molecules into micelles, which are formed in the solution of the glycyrrhizic acid disodium salt. Using the opisthorchiasis model caused by Opisthorchis felineus, we found a 4- to 11-fold increase in the anthelmintic activity of praziquantel in the composition as compared to the official praziquantel. According to the pharmacokinetic data, the use of the composition increased the bioavailability of praziquantel 3 times.

Natural Poly- and Oligosaccharides as Novel Delivery Systems for Plant Protection Compounds.

To increase the bioavailability of plant protection products, we have applied a new approach based on noncovalent association with natural water-soluble polysaccharides and oligosaccharides as delivery systems (DSs). The mechanochemical technique has been applied to prepare the solid-state nanodispersed compositions of antidote 1,8-naphthalic anhydride (NA) with arabinogalactan, sodium salt of carboxymethylcellulose, and glycyrrhizin as DSs. The effect of DSs on the solubility and the penetration of NA into the seeds of barley and wheat has been investigated by various physicochemical techniques. All DSs considerably enhance the solubility of NA and improve its penetration into the grain. The influence of polysaccharides and oligosaccharides on artificial lipid membranes was studied by the NMR relaxation method. It was concluded that the effect of polysaccharides and oligosaccharides on the penetration efficacy of plant protection products might be associated with the detected solubility enhancement and the affinity of DSs to the surface of cell membranes.

Spectroscopic and molecular dynamics characterization of glycyrrhizin membrane-modifying activity.

Glycyrrhizic acid (GA) is a triterpene glycoside extracted from licorice root. Due to its amphiphilicity GA is capable of forming complexes with a variety of hydrophobic molecules, substantially increasing their solubility. GA can enhance the therapeutic effects of various drugs. It was hypothesized that the increased bioavailability of the drug by GA is not only due to increased solubility, but also to enhancement of drug permeability through cell membranes. In this study the interaction of GA with POPC liposomes and model DOPC, POPC and DPPC bilayers was investigated by NMR with addition of shift reagents and MD simulations. This work helps to better understand the mechanism of enhanced drug bioavailability in the presence of GA. NMR and MD reveal that GA does penetrate into the lipid bilayer. NMR shows that GA changes the mobility of lipids. GA is predominantly located in the outer "half-layer" of the liposome and that the middle of the hydrophobic tails is the preferred location. GA freely passes through the bilayer surface to the inner part bringing a few water molecules. Also both approaches indicate pore formation in the presence of GA. The GA interaction with membranes is an additional aspect of the biological activity of GA-based drug delivery systems.

Low field photo-CIDNP in the intramolecular electron transfer of naproxen-pyrrolidine dyads.

Photoinduced processes with partial (exciplex) and full charge transfer in donor-acceptor systems are of interest because they are frequently used for modeling drug-protein binding. Low field photo-CIDNP (chemically induced dynamic nuclear polarization) for these processes in dyads, including the drug, (S)- and (R)-naproxen and (S)-N-methyl pyrrolidine in solutions with strong and weak permittivity have been measured. The dramatic influence of solvent permittivity on the field dependence of the N-methyl pyrrolidine (1)H CIDNP effects has been found. The field dependences of both (R,S)- and (S,S)-dyads in a polar medium are the curves with a single extremum in the area of the S-T+ terms intersection. Moreover, the CIDNP field dependences of the same protons measured in a low polar medium present curves with several extrema. The shapes of the experimental CIDNP field dependence with two extrema have been described using the Green function approach for the calculation of the CIDNP effects in the system without electron exchange interactions. The article discusses the possible causes of the differences between the CIDNP field dependence detected in a low-permittivity solvent with the strong Coulomb interactions and in a polar solvent.

Influence of glycyrrhizin on permeability and elasticity of cell membrane: perspectives for drugs delivery.

Glycyrrhizin or glycyrrhizic acid (GA) - triterpene glycoside extracted from licorice root - has been intensively studied over the past decade and is considered to be a potential drug delivery system. Glycyrrhizin was found to enhance the therapeutic effect of various drugs; however the detailed mechanism of these effects is still unknown and attracts the attention of researchers. In this work, we have made an attempt to clarify the mechanism of Glycyrrhizin activity on molecular and cellular level. The influence of GA on the functional properties of biomembranes was investigated via NMR spectroscopy and atomic force microscopy (AFM) using human erythrocytes as a model system. GA was shown to increase the permeability (about 60%) and to decrease elasticity modulus of cell membranes (by an order of magnitude) even in micromolar concentrations. Changes on the erythrocyte surface were also detected by AFM. These results could provide a new insight on the mechanism of bioavailability enhancement of some drugs in the presence of glycyrrhizin, as well as the mechanism of its own biological activity. The role of cholesterol-glycyrrhizin binding in the observed effects is also discussed.

Inhibition of Fe(2+)- and Fe(3+)- induced hydroxyl radical production by the iron-chelating drug deferiprone.

Deferiprone (L1) is an effective iron-chelating drug that is widely used for the treatment of iron-overload diseases. It is known that in aqueous solutions Fe(2+) and Fe(3+) ions can produce hydroxyl radicals via Fenton and photo-Fenton reactions. Although previous studies with Fe(2+) have reported ferroxidase activity by L1 followed by the formation of Fe(3+) chelate complexes and potential inhibition of Fenton reaction, no detailed data are available on the molecular antioxidant mechanisms involved. Similarly, in vitro studies have also shown that L1-Fe(3+) complexes exhibit intense absorption bands up to 800nm and might be potential sources of phototoxicity. In this study we have applied an EPR spin trapping technique to answer two questions: (1) does L1 inhibit the Fenton reaction catalyzed by Fe(2+) and Fe(3+) ions and (2) does UV-Vis irradiation of the L1-Fe(3+) complex result in the formation of reactive oxygen species. PBN and TMIO spin traps were used for detection of oxygen free radicals, and TEMP was used to trap singlet oxygen if it was formed via energy transfer from L1 in the triplet excited state. It was demonstrated that irradiation of Fe(3+) aqua complexes by UV and visible light in the presence of spin traps results in the appearance of an EPR signal of the OH spin adduct (TMIO-OH, a(N)=14.15G, a(H)=16.25G; PBN-OH, a(N)=16.0G, a(H)=2.7G). The presence of L1 completely inhibited the OH radical production. The mechanism of OH spin adduct formation was confirmed by the detection of methyl radicals in the presence of dimethyl sulfoxide. No formation of singlet oxygen was detected under irradiation of L1 or its iron complexes. Furthermore, the interaction of L1 with Fe(2+) ions completely inhibited hydroxyl radical production in the presence of hydrogen peroxide. These findings confirm an antioxidant targeting potential of L1 in diseases related to oxidative damage.

Antioxidant and redox properties of supramolecular complexes of carotenoids with beta-glycyrrhizic acid.

Supramolecular complexes between carotenoids and a triterpene glycoside, beta-glycyrrhizic acid (GA), were found to exhibit unusual antioxidant activity. Complexation with GA increases a scavenging rate of canthaxanthin and 7',7'-dicyano-7'-apo-beta-carotene toward OOH radicals more than 10 times, but has no effect on the scavenging rate of zeaxanthin. Scavenging rate constants were measured in DMSO solution of carotenoids using the EPR spin-trapping technique. EPR parameters of spin adducts were determined as a(H) = 2.3 G, a(N) = 13.9 G for PBN (N-tert-butyl-alpha-phenylnitrone)-OOH, and a(H) = 3.4 G, a(N) = 14.9 G for the PBN-CH3 adduct. Taking into account the previously measured dependence of the scavenging rate constants toward OOH radicals on the oxidation potential of carotenoids, this result can be explained by the hypothesis that the complexation with GA affects the value of oxidation potentials. This hypothesis was confirmed by CV measurements.

Carotenoids as scavengers of free radicals in a Fenton reaction: antioxidants or pro-oxidants?

The spin trapping EPR technique was used to study the influence of carotenoids (beta-carotene, 8'-apo-beta-caroten-8'-al, canthaxanthin, and ethyl 8'-apo-beta-caroten-8'-oate) on the yield of free radicals in the Fenton reaction (Fe(2+) + H(2)O(2) --> Fe(3+) + .OH + -OH) in the organic solvents, DMSO, and methanol. DMPO and PBN were used as spin trapping agents. It was demonstrated that carotenoids could increase or decrease the total yield of free radicals depending on the oxidation potential of the carotenoids and the nature of the radicals. A reaction mechanism is suggested which includes the reduction of Fe(3+) to Fe(2+) by carotenoids. The effectiveness of this carotenoid-driven Fenton reaction increases with a decrease of the scavenging rates for free radicals and with decreasing oxidation potentials of carotenoids.

Carotenoids as antioxidants: spin trapping EPR and optical study.

The role of several natural and synthetic carotenoids as scavengers of free radicals was studied in homogeneous solutions. A set of free radicals: *OH, *OOH, and *CH(3) were generated by using the Fenton reaction in dimethyl sulfoxide. It was shown that the spin trapping technique is more informative than optical methods for the experimental conditions under study. 5,5-Dimethyl-pyrroline-N-oxide (DMPO) and N-tert-butyl-alpha-phenylnitrone (PBN) were used as spin traps for the EPR studies. The results show that the scavenging ability of the carotenoids towards radical *OOH correlates with their redox properties.

EPR spin trapping detection of carbon-centered carotenoid and beta-ionone radicals.

Free radical intermediates were detected by the electron paramagnetic resonance spin trapping technique upon protonation/deprotonation reactions of carotenoid and beta-ionone radical ions. The hyperfine coupling constants of their spin adducts obtained by spectral simulation indicate that carbon-centered radicals were trapped. The formation of these species was shown to be a result of chemical oxidation of neutral compounds by Fe(3+) or I(2) followed by deprotonation of the corresponding radical cations or addition of nucleophilic agents to them. Bulk electrolysis reduction of beta-ionone and carotenoids also leads to the formation of free radicals via protonation of the radical anions. Two different spin adducts were detected in the reaction of carotenoid polyenes with piperidine in the presence of 2-methyl-2-nitroso-propane (MNP). One is attributable to piperidine radicals (C(5)H(10)N*) trapped by MNP and the other was identified as trapped neutral carotenoid (beta-ionone) radical produced via protonation of the radical anion. Formation of these radical anions was confirmed by ultraviolet-visible spectroscopy. It was found that the ability of carotenoid radical anions/cations to produce neutral radicals via protonation/deprotonation is more pronounced for unsymmetrical carotenoids with terminal electron-withdrawing groups. This effect was confirmed by the radical cation deprotonation energy (H(D)) estimated by semiempirical calculations. The results indicate that the ability of carotenoid radical cations to deprotonate decreases in the sequence: beta-ionone > unsymmetrical carotenoids > symmetrical carotenoids. The minimum H(D) values were obtained for proton abstraction from the C(4) atom and the C(5)-methyl group of the cyclohexene ring. It was assumed that deprotonation reaction occurs preferentially at these positions.

Photochemistry of acetylenic ketones in micellar solutions as studied by product-yield-detected ESR and transient absorption techniques.

The electron spin resonance (ESR) spectra of the transient radical pairs in the photoreduction of 1,5-diphenyl-1,4-pentadiyn-3-one(I) and 1,3-diphenyl-2-propyn-1-one(II) in sodium dodecyl sulfate (SDS) micellar solutions have been obtained by using the product-yield-detected ESR (PYESR) technique. The PYESR spectra, detected by tracing the microwave effect on the spin-adduct yield as functions of the magnetic field, show the ESR spectra of the ketyl radical of the ketone and SDS radical as the components of the radical pairs. In addition, the growth and the decay processes of the radical pair were observed through detecting the effect of microwave pulse as functions of the delay period between a laser pulse and the off and on time, respectively, of a microwave pulse. The absorption spectra of transient species have also been obtained by using the laser flash photolysis technique. Through the analysis of these data and molecular orbital calculations, the role of acetylenic groups in the photoreactivity of acetylenic ketones is discussed.