The Endless World of Carotenoids-Structural, Chemical and Biological Aspects of Some Rare Carotenoids.

Carotenoids are a large and diverse group of compounds that have been shown to have a wide range of potential health benefits. While some carotenoids have been extensively studied, many others have not received as much attention. Studying the physicochemical properties of carotenoids using electron paramagnetic resonance (EPR) and density functional theory (DFT) helped us understand their chemical structure and how they interact with other molecules in different environments.

Carotenoids: Importance in Daily Life-Insight Gained from EPR and ENDOR.

Carotenoids are indispensable molecules for life. They are present everywhere in plants, algae, bacteria whom they protect against free radicals and oxidative stress. Through the consumption of fruits and vegetables and some carotenoid-containing fish, they are introduced into the human body and, similarly, protect it.

Antioxidant Activity in Supramolecular Carotenoid Complexes Favored by Nonpolar Environment and Disfavored by Hydrogen Bonding.

Carotenoids are well-known antioxidants. They have the ability to quench singlet oxygen and scavenge toxic free radicals preventing or reducing damage to living cells. We have found that carotenoids exhibit scavenging ability towards free radicals that increases nearly exponentially with increasing the carotenoid oxidation potential.

Supramolecular Carotenoid Complexes of Enhanced Solubility and Stability-The Way of Bioavailability Improvement.

Carotenoids are natural dyes and antioxidants widely used in food processing and in therapeutic formulations. However, their practical application is restricted by their high sensitivity to external factors such as heat, light, oxygen, metal ions and processing conditions, as well as by extremely low water solubility. Various approaches have been developed to overcome these problems.

Photo-induced charge separation in hydroxycoumarins on TiO and F-TiO.

The efficiency of photocatalytic charge separation is much higher for 7-hydroxycoumarin (7-CN) and 6,7-dihydroxycoumarin (6,7-CN) adsorbed on the surface modified TiO where the surface hydroxyl group was replaced by a fluorine atom (F-TiO) than on TiO. EPR measurements find 5- and 12-fold increases in free radical yields for 7-CN and 6,7-CN, respectively. DFT calculations for the coumarins on TiO and F-TiO were performed to investigate these phenomena.

The carotenoid Bixin found to exhibit the highest measured carotenoid oxidation potential to date consistent with its practical protective use in cosmetics, drugs and food.

The electrochemical oxidation potentials of cis bixin correspond to the production of the carotenoid radical cation, Car and dication Car. The oxidation is a two-electron process with oxidation potentials at ~0.94 and ~1.

Photo Protection of Haematococcus pluvialis Algae by Astaxanthin: Unique Properties of Astaxanthin Deduced by EPR, Optical and Electrochemical Studies.

The antioxidant astaxanthin is known to accumulate in algae under unfavorable environmental conditions for normal cell growth. The accumulated astaxanthin functions as a protective agent against oxidative stress damage, and tolerance to excessive reactive oxygen species (ROS) is greater in astaxanthin-rich cells. The detailed mechanisms of protection have remained elusive, however, our Electron Paramagnetic Resonance (EPR), optical and electrochemical studies on carotenoids suggest that astaxanthin's efficiency as a protective agent could be related to its ability to form chelate complexes with metals and to be esterified, its inability to aggregate in the ester form, its high oxidation potential and the ability to form proton loss neutral radicals under high illumination in the presence of metal ions.

Radicals formed from proton loss of carotenoid radical cations: A special form of carotenoid neutral radical occurring in photoprotection.

In an organized assembly in Arabidopsis thaliana plant, proton loss from the radical cation of zeaxanthin (Zea) was found to occur under intense illumination, a possible component in photoprotection. A stable neutral radical is formed because of the favorable proton loss at C4(4') position(s) of the terminal ends of Zea that extends the unpaired spin density distribution (notation Zea(4) or Zea(4') by symmetry). Proton loss from the radical cation of β-carotene (β-car) to available proton acceptors was also detected in a PSII sample upon irradiation.

Hydrogen Bond Formation between the Carotenoid Canthaxanthin and the Silanol Group on MCM-41 Surface.

The formation of one or two hydrogen bonds (H-bonds) between canthaxanthin (CAN), a dye, and the silanol group(s) on the MCM-41 surface has been studied by density functional theory (DFT) calculations and calorimetric experiments. It was found that the formation of the H-bond(s) stabilized the CAN molecule more than its radical cation (CAN(•+)). The charge distribution, bond lengths, and the HOMO and LUMO energies of CAN are also affected.

Water soluble biocompatible vesicles based on polysaccharides and oligosaccharides inclusion complexes for carotenoid delivery.

Since carotenoids are highly hydrophobic, air- and light-sensitive hydrocarbon compounds, developing methods for increasing their bioavailability and stability towards irradiation and reactive oxygen species is an important goal. Application of inclusion complexes of "host-guest" type with polysaccharides and oligosaccharides such as arabinogalactan, cyclodextrins and glycyrrhizin minimizes the disadvantages of carotenoids when these compounds are used in food processing (colors and antioxidant capacity) as well as for production of therapeutic formulations. Cyclodextrin complexes which have been used demonstrated enhanced storage stability but suffered from poor solubility.

Chemistry of carotenoid neutral radicals.

Proton loss from the carotenoid radical cations (Car(+)) to form neutral radicals (#Car) was investigated by numerous electrochemical, EPR, ENDOR and DFT studies described herein. The radical cation and neutral radicals were formed in solution electrochemically and stabilized on solid silica-alumina and MCM-41 matrices. Carotenoid neutral radicals were recently identified in Arabidopsis thaliana plant and photosystem II samples.

DFT and ENDOR Study of Bixin Radical Cations and Neutral Radicals on Silica-Alumina.

Bixin, a carotenoid found in annatto (Bixa orellana), is unique among natural carotenoids by being water-soluble. We stabilized free radicals from bixin on the surface of silica-alumina (Si-Al) and characterized them by pulsed electron-nuclear double resonance (ENDOR). DFT calculations of unpaired electron spin distribution for various bixin radicals predict the EPR hyperfine couplings.

A DFT study of the interaction between olefins and Cu2+ on silica and MCM-41 model surfaces.

The interaction between ethylene and Cu(2+) on a silica model surface was studied by density functional theory (DFT) with nine popular functionals. It is found that B3LYP with BSSE correction is the best method by comparing the calculated results with reported experimental data. This method was also used to study the interactions of Cu(2+) with β-carotene, 1,3,5,7,9,11,13-tetradecaheptaene and ethylene on a MCM-41 model surface.

Electrochemical study of astaxanthin and astaxanthin n-octanoic monoester and diester: tendency to form radicals.

The carotenoid astaxanthin known for its powerful antioxidant activity was electrochemically investigated along with the synthesized astaxanthin n-octanoic monoester and astaxanthin n-octanoic diester. Cyclic voltammograms (CVs) revealed a two-electron transfer oxidation for all three carotenoids with a difference in the two oxidation potentials (ΔE = E2(0) - E1(0)) slightly increasing from astaxanthin to the monoester to diester. Minimal or no exposure to water prevented the formation of carotenoid neutral radicals from dications and radical cations, causing near absence of the fifth peak in the CVs.

Photochemical and optical properties of water-soluble xanthophyll antioxidants: aggregation vs complexation.

Xanthophyll carotenoids can self-assemble in aqueous solution to form J- and H-type aggregates. This feature significantly changes the photophysical and optical properties of these carotenoids, and has an impact on solar energy conversion and light induced oxidative damage. In this study we have applied EPR and optical absorption spectroscopy to investigate how complexation can affect the aggregation ability of the xanthophyll carotenoids zeaxanthin, lutein, and astaxanthin, their photostability, and antioxidant activity.

Ethers and esters derived from apocynin avoid the interaction between p47phox and p22phox subunits of NADPH oxidase: evaluation in vitro and in silico.

NOX (NADPH oxidase) plays an important role during several pathologies because it produces the superoxide anion (O2•-), which reacts with NO (nitric oxide), diminishing its vasodilator effect. Although different isoforms of NOX are expressed in ECs (endothelial cells) of blood vessels, the NOX2 isoform has been considered the principal therapeutic target for vascular diseases because it can be up-regulated by inhibiting the interaction between its p47phox (cytosolic protein) and p22phox (transmembrane protein) subunits. In this research, two ethers, 4-(4-acetyl-2-methoxy-phenoxy)-acetic acid (1) and 4-(4-acetyl-2-methoxy-phenoxy)-butyric acid (2) and two esters, pentanedioic acid mono-(4-acetyl-2-methoxy-phenyl) ester (3) and heptanedioic acid mono-(4-acetyl-2-methoxy-phenyl) ester (4), which are apocynin derivatives were designed, synthesized and evaluated as NOX inhibitors by quantifying O2•- production using EPR (electron paramagnetic resonance) measurements.

Neutral carotenoid radicals in photoprotection of wild-type Arabidopsis thaliana.

The deprotonation of naturally occurring zeaxanthin (Zea) radical cations (Zea(•+)) to form neutral radicals (#Zea(•)) and their involvement in the qE portion of nonphotochemical quenching (NPQ) was examined. The radical cations are weak acids, and readily deprotonate to a long-lived neutral radical (#Zea(•)) that could serve as long-lived quenching sites. When #Zea(•) is eventually neutralized and Zea is reformed in the presence of D2O, the Zea has an opportunity to undergo H/D exchange.

EPR study of the astaxanthin n-octanoic acid monoester and diester radicals on silica-alumina.

The radical intermediates of the n-octanoic monoester and n-octanoic diester of astaxanthin were detected by pulsed EPR measurements carried out on the UV-produced radicals on silica-alumina artificial matrix and characterized by density functional theory (DFT) calculations. Previous Mims ENDOR for astaxanthin detected the radical cation and neutral radicals formed by proton loss from the C3 (or C3') position and from the methyl groups. Deprotonation of the astaxanthin neutral radical formed at the C3 (or C3') position resulted in a radical anion.

Carotenoid radical formation: dependence on conjugation length.

The relative energy of carotenoid neutral radicals formed by proton loss from the radical cations of linear carotenoids has been examined as a function of conjugation length from n = 15 to 9. For a maximum conjugation length of n = 15 (bisdehydrolycopene, a symmetrical compound), proton loss can occur from any of the 10 methyl groups, with proton loss from the methyl group at position C1 or C1' being the most favorable. In contrast, the most energetically favorable proton loss from the radical cations of lycopene, neurosporene, spheroidene, spheroidenone, spirilloxanthin, and anhydrorhodovibrin occurs from methylene groups that extend from the conjugated system.

Free radical formation in novel carotenoid metal ion complexes of astaxanthin.

The carotenoid astaxanthin forms novel metal ion complexes with Ca(2+), Zn(2+), and Fe(2+). MS and NMR measurements indicate that the two oxygen atoms on the terminal cyclohexene ring of astaxanthin chelate the metal to form 1:1 complexes with Ca(2+) and Zn(2+) at low salt concentrations <0.2 mM.

CIDNP and EPR study of phototransformation of lappaconitine derivatives in solution.

Chemically induced dynamic nuclear polarization (CIDNP) and electron paramagnetic resonance (EPR) techniques have been used to study the paramagnetic species formed during the photolysis of the alkaloid lappaconitine and its synthetic analogues in solution. Lappaconitine is a photosensitive antiarrhythmic and hypertension drug, whose major photoproduct (N-acetyl anthranilic acid) is also a potent photosensitizer. Both these compounds are lipophilic and might bind efficiently to cell membranes thereby causing phototoxic damage.

Enhancement of the photocatalytic activity of TiO2 nanoparticles by water-soluble complexes of carotenoids.

Photoirradiation of TiO(2) nanoparticles by visible light in the presence of the water-soluble natural polysaccharide arabinogalactan complexes of the hydrocarbon carotenoid β-carotene leads to enhanced yield of the reactive hydroxyl (OH) radicals. The electron paramagnetic resonance (EPR) spin-trapping technique using α-phenyl-N-tert-butyl nitrone (PBN) as the spin-trap has been applied to detect this intermediate by trapping the methyl and methoxy radicals generated upon reaction of the hydroxyl radical with dimethylsulfoxide (DMSO). The free radicals formed in this system proceed via oxygen reduction and not via the reaction of holes on the TiO(2) surface.

Formation of carotenoid neutral radicals in photosystem II.

beta-Carotene radicals produced in the hexagonal pores of the molecular sieve Cu(II)-MCM-41 were studied by ENDOR and visible/near-IR spectroscopies. ENDOR studies showed that neutral radicals of beta-carotene were produced in humid air under ambient fluorescent light. The maximum absorption wavelengths of the neutral radicals were measured and were additionally predicted by using time-dependent density functional theory (TD-DFT) calculations.

Measuring Ti(III)-carotenoid radical interspin distances in TiMCM-41 by pulsed EPR relaxation enhancement method.

Interspin distances between the Ti(3+) ions and the carotenoid radicals produced inside TiMCM-41 pores by photoinduced electron transfer from 7'-apo-7'-(4-carboxyphenyl)-beta-carotene (coordinated to Ti(3+)), canthaxanthin (formed as a random distribution of isomers), and beta-ionone (model for a short-chain polyene) to Ti(3+) framework sites were determined using the pulsed EPR relaxation enhancement method. To estimate the electron transfer distances, the temperature dependence of relaxation rates was analyzed in both siliceous and metal-substituted siliceous materials. The phase memory times, T(M), of the carotenoid radicals were determined from the best fits of two-pulse ESEEM curves.

Structure and properties of 9'-cis neoxanthin carotenoid radicals by electron paramagnetic resonance measurements and density functional theory calculations: present in LHC II?

The radical intermediates formed upon catalytic or photooxidation of the carotenoid 9'-cis neoxanthin inside MCM-41 molecular sieves were detected by pulsed Mims and Davies electron nuclear double resonance (ENDOR) spectroscopies and characterized by density functional theory (DFT) calculations. Mims ENDOR spectra (20 K) were simulated using the hyperfine coupling constants predicted by DFT, which showed that a mixture of carotenoid radical cations (Car(+)) and neutral radicals (#Car) is formed. The DFT relative energies of the neutral radicals formed by proton loss from the C5, C5', C9, C9', C13, and C13'-methyl groups of Car(+) showed that #Car(9') is energetically most favorable, while #Car(9), #Car(13), #Car(13'), #Car(5'), and #Car(5) are less favorable for formation by 2.