Research paperThe effect of polarity of environment on the antioxidant activity of carotenoids
Introduction
Carotenoids are tetraterpene pigments that are distributed in photosynthetic bacteria, some species of archaea and fungi, algae, plants, and animals. They are essential pigments in photosynthetic organs along with chlorophylls. They also act as photo-protectors, antioxidants, color attractants, and precursors of plant hormones in non-photosynthetic organs of plants [1]. Carotenoids are ubiquitous and essential pigments in photosynthesis. They absorb in the blue-green region of the solar spectrum and transfer the absorbed energy to (bacterio-)chlorophylls, and so expand the wavelength range of light that is able to drive photosynthesis [2]. Carotenoids frequently occur in vivo in association with proteins. Carotenoid-protein complexes have been isolated from many sources, e.g. photosynthetic membranes. The most striking and intriguing are the carotenoproteins, from marine invertebrate animals, in which binding to protein causes a large shift in the carotenoid light absorption spectrum so that the complexes show different colours [3]. The orange carotenoid protein (OCP) is a water-soluble, photoactive protein involved in thermal dissipation of excess energy absorbed by the light-harvesting phycobilisomes (PBS) in cyanobacteria [4], [5]. OCP is a possible system to increase the water solubility of carotenoids [5]. The important role of carotenoids in carotenoid-chloprophyll proteins has also been studied [6].
Carotenoids are natural dyes and antioxidants widely used in food processing and in therapeutic formulations due to their abilities to react with toxic free radicals to prevent damage to living cells [7], [8], [9]. Diseases such as infarction, cerebral thrombosis and tumors are partly a result of the action of free radicals and reactive oxygen species (ROS) [10]. It is known that the most of adsorbed oxygen in the lungs is used for energy production, but about 1–3% of this oxygen is used to make harmful ROS, such as hydrogen peroxide (H2O2) and the superoxide radical (O2
−) [10]. When these ROS react with transition metals (e.g., iron and copper), very reactive free radical species such as hydroxyl (OH) radicals are produced. The OH radical poses major threat as it is able to destroy almost every cell in the human body [10], [11].
It is reported that carotenoids exhibit scavenging ability towards the free radicals which increases nearly exponentially with increasing carotenoid oxidation potential [12], [13]. For example, bixin exhibits the highest measured carotenoid oxidation potential (0.94 V vs SCE) to date [14]. The scavenging ability of cis-bixin towards ROS such as OH, OOH and O2− was estimated to be 17 times higher than that of carotenoid astaxanthin with an oxidation potential of 0.768 V, and 69 times higher than that of carotenoid β–carotene with an oxidation potential of 0.60 V [14]. Because of its low oxidation potential, β–carotene even exhibits a prooxidant behavior. For example, the radical of β–carotene is formed when β–carotene reacts with Fe3+ [13].
Hydrophobicity of carotenoids restricts wide practical applications with physiological benefits. Carotenoids also possess chemical instability because the polyene chains of carotenoids are long with up to 15 conjugated double bonds. This chemical feature is responsible for their instability to light, high temperature, oxygen and metal ions or interactions with radical species [15], [16]. It was determined that low photostability of carotenoids is related to the formation of carotenoid radical cations that form when electrons are being transferred to acceptors [17]. A radical cation can lose a proton to form a neutral radical in the presence of water or other proton acceptors. This neutral radical is a highly reactive species, which can form a set of oxidation products and carotenoid dimers [18]. Additionally, reactions of carotenoids with ROS and metal ions, such as Fe3+ and Cu2+, inside the human body accelerate the metabolism of carotenoids reducing their bioavailability [19].
Therefore, it is necessary to develop methods for increasing bioavailability of carotenoids and stability towards irradiation and ROS. For this purpose, carotenoids have been incorporated into host molecules such as cyclodextrins (CD) [20], [21], arabinogalactan (AG) [22] and β-glycyrrhizic acid (GA) [9], [23], [24] lipid nanoparticles [25] and MCM-41[26]. The techniques for the incorporation include the traditional “liquid phase” techniques and the solid state techniques, and the details are described in a recent review article [19]. It was found that the incorporation of carotenoids in host molecules enhanced water solubility and oxidation stability of carotenoids. The complexes of carotenoids with AG have shown enhanced photostability by a factor of 10 in water solutions, and a significant decrease by a factor of 20 in the reactivity towards metal ions (Fe3+) and ROS in solution [22]. The complexation with GA increased the oxidation potentials, which resulted in increased antioxidant activity of selected carotenoids [9], [27]. Other benefits of the incorporation include prevention of aggregation of xanthophyll carotenoids in aqueous solutions, and a remarkable increase in the quantum yield and the lifetime of the charge-separated states of the carotenoid radical cations [28]. Encapsulation of lipophilic carotenoids with different delivery systems is an innovative approach shown to increase their solubility, stability, bioavailability and controlled release in human body [29], [30]. Different delivery systems such as inclusion complexes, nanoemulsions, nanoliposomes, and biopolymeric nanoparticles have been tested to improve carotenoids’ properties. The encapsulation methods vary with different delivery systems and carotenoids, and are reviewed in Ref. [19].
As mentioned above, the scavenging ability of carotenoids towards free radicals is strongly potential dependent [12], [13]. It was demonstrated that GA complexation can affect the oxidation potential of the carotenoids. For example, the oxidation potentials of two carotenoids, zeaxanthin and canthaxanthin, in the presence of GA increase by 0.03–0.05 V compared with those in dimethyl sulfoxide (DMSO) [9], [27]. The scavenging rate constant of OOH radicals by GA complex is much larger than the free carotenoid (e.g., 59 vs 2 for canthaxanthin) [9], [27]. The encapsulation of carotenoids in different delivery systems also enhanced the antioxidant activity in different antioxidant models [25], [31]. The antioxidant activity of carotenoids after being encapsulated in liposomes was in the order lutein > β-carotene > lycopene > canthaxanthin [25].
It is important to know how different environments affect the oxidation potentials of carotenoids and thus their antioxidant activities. The incorporation of carotenoids into “hosts” results in noncovalent binding (hydrophobic forces, van der Waals interactions or hydrogen bonds) between the nonpolar carotenoid and the “hosts” [23]. Thus, the polarity of a host affects the stabilities of the neutral species and those of the radical cations, which in turn affects the oxidation potentials of carotenoids. The purpose of this study is to determine how polarities of different environments affect the oxidation potentials of carotenoids via DFT calculations. The polarities of the environments can be simulated by adding the carotenoids in solvents with different polarities. By comparing the calculated oxidation potentials of carotenoids in the different solvents, one can determine the effect of polarity of different environments on the oxidation potential of the carotenoid. The results of this study will be beneficial in improving the design of the supramolecular carotenoid complexes.
Section snippets
Computational methods
For the reduction of a carotenoid radical cation (Car+) to the carotenoid neutral species (Car) in a solvent(s):Car+(s) + e− → Car(s)
Car+(s) and Car(s) represent Car+ and Car in a solvent, respectively. The half-cell standard redox potentials were obtained by computing the Gibbs free energy change △Gred(s) due to the reduction of Car+ in the solution, as follows:with the Faraday constant F = 23.06 kcal mol−1 V−1 and n = 1 the number of electrons involved in the redox couple. The
Computational details
All computations were performed with the Gaussian 09 [33] suite of programs. To calculate the Gibbs free energies of the neutral species I and the radical cation of I (I+) in the gas phase, the structures of I and I+ were optimized with the default convergence. Four density functionals (DFs) B3LYP [35], PBE1PBE [36], M06-2X [37] and wB97XD [38] were chosen in the calculations respectively. For B3LYP, PBE1PBE and M06-2X, dispersion effects were taken into account from Grimme’s empirical
Determination of oxidation potentials of carotenoids from cyclic voltammetry (CV)
The CV data for the oxidation of I and IV in CH2Cl2 with Fc/Fc+ (Fc = ferrocene) as reference are available in the study by Tan et al. [34], and these experimental data were compared to the calculated values by the DFT calculations. The CV for the oxidation of I in CH2Cl2 is irreversible with the absence of the cathodic peak in the reverse scan. According to a recent study [42], the half-wave potentials, which are readily obtainable from cyclic voltammograms, are by far the best representation
Results and discussion
Since I is much smaller in size (contains less atoms) than IV, the calculations of I are much cheaper than those of IV. Therefore, the experimental oxidation potential of I was used as the benchmark in the DFT calculations to determine which method is the best. The calculated Gibbs free energy changes in the gas phase △Gred(g) (kcal/mol) with four DFs and two BSs are listed in Table 2. M06-2X + D3 gives values that are much more negative than those calculated with B3LYP + D3, PBBE1PBE + D3 and
Conclusions
The DFT calculations with the density functional M06-2X + D3 and the C-PCM continuum solvation model provide accurate prediction of the oxidation potentials of carotenoids in solvents with different polarities. The oxidation potential of a carotenoid increases with the decrease of the polarity of a solvent. The difference in the oxidation potential of a carotenoid in a non polar solvent with ε of about 2 versus that in a very polar solvent with ε of about 80, can be as large as 0.6 V. These
CRediT authorship contribution statement
Yunlong Gao: Conceptualization, Methodology, Investigation, Writing - original draft. A. Ligia Focsan: Writing - review & editing. Lowell D. Kispert: Writing - review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was financially supported by the Faculty Research Rewards Program at the Nanjing Agricultural University.
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