Neutral carotenoids

Goldie, A.H. and Subden, R.E., The neutral carotenoids of wild-type and mutant strains of Neurospora crassa, Biochem. Genet. 10, 275, 1973. 

The ferric ion is often used to form the carotenoid radical cation. However, care must be taken to control the concentration of the ferric ion relative to that of the carotenoid. Several existing equilibria have been studied by EPR, as well as NMR, LC-MS, and optical techniques. These studies have shown the following equilibria (Scheme 9.2) depending on the concentrations of Fe3+, Fe2+, and Ck relative to that of the neutral carotenoid and its radical cation and dication. 

Using different DFT functionals and basis sets (Focsan et al. 2008, Lawrence et al. 2008) it was confirmed that the isotropic ()-methyl proton hyperfine couplings do not exceed 9MHz for the carotenoid radical cation, Car-. DFT calculations of neutral carotenoid radicals, Car formed by proton loss (indicated by ) from the radical cation, predicted isotropic P-methyl proton couplings up to 16 MHz, a fact that explained the large isotropic couplings observed by ENDOR measurements for methyl protons in UV irradiated carotenoids supported on silica gel, Nafion films, silica-alumina matrices, or incorporated in molecular sieves (Piekara-Sady et al. 1991, 1995, Wu et al. 

Skibsted and coworkers (Mortensen and Skibsted 1996) have shown that upon the laser flash photolysis of carotenoids in chloroform bleaching of the ground state absorption is observed and there is formation of two near infrared-absorbing species ()tmax 920 and lOOOnm for 0-CAR). The species absorbing at about lOOOnm is 0-CAR + and, as with the carotenoid/CCl302 system noted earlier, the 0-CAR,+ is formed from the other species. The nature of the other species is not defined although an adduct or a neutral carotenoid radical is proposed. 

Straub, O. Key to Carotenoids List of Neutral Carotenoids, Birkhauser-Verlag, Basel, Stuttgart, 1976... 

The electrochemical properties of carotenoids has been reviewed [102,103]. Electrochemical data for several naturally occurring carotenoids including oxidation potentials, reaction rate constants and kinetic equilibrium constants were discussed. Conventional electrochemical techniques such as cyclic voltammetry (CV) were treated. The oxidation potential of neutral carotenoids, corresponding to the formation of cation radicals were 0.50 - 0.72 V versus SCE, and that of carotenoid cation radicals, corresponding to the formation of dications were 0.52 - 0.95 V versus SCE. Further details are available [104,105],... 

The principles, scope and limitations of Raman spectroscopy and its application in the carotenoid field has been discussed [134], Time-resolved resonance Raman spectroscopy provides useful information on carotenoids in the excited state [135]. Differences between the resonance Raman spectra of a neutral carotenoid and its cation radical provide structural information on the cation radical. 

An intermediate role of cation radicals in cis-trans isomerisation of carotenoids has been considered [138]. AMI molecular orbital calculations show that the energy barrier of cis-trans isomerisation are much lower in cation radicals ( 20 kcal/mol) and dications ( 0 kcal/mol) than in neutral carotenoids ( 55 kcal/mol). HPLC analyses of the product mixture after bulk electrolysis of [3-carotene (1), canthaxanthin (16) and apocarotenoids showed the presence of 5-cis, 13-cis, 9-cis, 9,13-dicz s and all-trans isomers [138]. 

Neutral carotenoid Cation radical Dication Car Car+ Car2+... 

The equilibrium between neutral carotenoid, cation radical and dication was already discussed [142,143], More recently the effect of electrolytes and temperature on carotenoid dications were studied [40]. The stability of the P-carotene (1) dication at -25°C in CHCI3 was remarkable, showing a decrease of less than 20% during 2h, as based on NIR spectra [11]. 

It was observed that electrochemical oxidation of all-trans P-carotene (1) and canthaxanthin (16) in CH2CI2 leads to significant trans-cis isomerisation [105]. It was suggested that the isomerisation mechanism involved cation radicals and/or dications which could easily undergo geometrical isomerisation. This proposal was supported by AMI molecular orbital calculations, which showed that the energy barrier from trans to cis is much lower in the cation radical and dication species than in the neutral carotenoid [105]. 

Electrochemical and Electron paramagnetic resonance (EPR) studies have been focused on neutral carotenoid radicals. These species are derived from carotenoid radical cations deprotonation (Focsan et al., 2015). At lower radical concentration this behaves like a scavenger for reactive species (single oxygen, hydroxyl or peroxyl radicals) (Foot, 1976). Although the antioxidant activity of carotenoids is higher than that of a-tocopherol, a modest contribution (less than 5%, due to modest concentration levels of carotenoids in oil) to the total activity is expected (Muller et al., 2011). 

An open column packed with neutral aluminium oxide (grade III) slurry is generally used for semi-preparative separation of large amounts of carotenoid extract, revealing three broad bands (1) carotenes and epoxy-carotenes constitute the first fraction to elute with petroleum ether, (2) monohydroxy and keto-carotenoids with 50 to 80% diethyl ether in petroleum ether are next, and (3) finally, the polyhydroxy carotenoids elute with 2 to 5% diethyl ether in ethanol or... 

Capillary electrophoresis (CE) has several unique advantages compared to HPLC, snch as higher efficiency dne to non-parabolic fronting, shorter analytical time, prodnction of no or much smaller amounts of organic solvents, and lower cost for capillary zone electrophoresis (CZE) and fused-silica capillary techniques. However, in CZE, the most popular separation mode for CE, the analytes are separated on the basis of differences in charge and molecular sizes, and therefore neutral compounds snch as carotenoids do not migrate and all co-elute with the electro-osmotic flow. 

APCl in positive mode ionization and triple quadrupole detection was used for determination of free and bound carotenoids in paprika, obtaining the [M + H]+ and losses of fatty acids as neutral molecules from the [M + H]+ with MeOH, MTBE, and H2O as eluent from the C30 column. The positions of the fatty acids on unsymmetrical xanthophylls could not be established by the MS data. 

Other dietary factors implicated in prostate cancer include retinol, carotenoids, lycopene, and vitamin D consumption.5,6 Retinol, or vitamin A, intake, especially in men older than age 70, is correlated with an increased risk of prostate cancer, whereas intake of its precursor, [3-carotene, has a protective or neutral effect. Lycopene, obtained primarily from tomatoes, decreases the risk of prostate cancer in small cohort studies. The antioxidant vitamin E also may decrease the risk of prostate cancer. Men who developed prostate cancer in one cohort study had lower levels of l,25(OH)2-vitamin D than matched controls, although a prospective study did not support this.2 Clearly, dietary risk factors require further evaluation, but because fat and vitamins are modifiable risk factors, dietary intervention may be promising in prostate cancer prevention. 

In this chapter, various EPR techniques that have been used to study the carotenoid radical cations and neutral radicals will be described. These methods with references are given in Table 9.1. 

Comproportionation equilibrium constants for Equation 9.3 between dications and neutral molecules of carotenoids were determined from the SEEPR measurements. It was confirmed that the oxidation of the carotenoids produced n-radical cations (Equations 9.1 and 9.3), dications (Equation 9.2), cations (Equation 9.4), and neutral ir-radicals (Equations 9.5 and 9.6) upon reduction of the cations. It was found that carotenoids with strong electron acceptor substituents like canthaxanthin exhibit large values of Kcom, on the order of 103, while carotenoids with electron donor substituents like (J-carotene exhibit Kcom, on the order of 1. Thus, upon oxidation 96% radical cations are formed for canthaxanthin, while 99.7% dications are formed for P-carotene. This is the reason that strong EPR signals in solution are observed during the electrochemical oxidation of canthaxanthin. 

Various EPR Techniques Used to Study Radical Cations and Neutral Radicals of Carotenoids... 

Konovalova and Kispert 1998, Konovalova et al. 1999, 2001a, Gao et al. 2002, 2003). It has now been shown that the carotenoid neutral radical is not formed in the absence of UV photolysis, and thus no resolvable methyl proton coupling of 13-16MHz would have been observed. 

CW ENDOR spectrum measurements carried out at 120 K (the optimum temperature for measuring resolved CW ENDOR powder spectra of carotenoid radicals) shows resolved lines from the P-methyl hfc (Piekara-Sady et al. 1991,1995, Wu et al. 1991, Jeevarajan et al. 1993b) (see Figure 9.5). The lines above 19 MHz are due to neutral radicals according to DFT calculations (Gao et al. 2006). 

Carotenoid neutral radicals are also formed under irradiation of carotenoids inside molecular sieves. Davies and Mims ENDOR spectra of lutein (Lut) radicals in Cu-MCM-41 were recorded and then compared with the simulated spectra using the isotropic and anisotropic hfcs predicted by DFT. The simulation of lutein radical cation, Lut +, generated the Mims ENDOR spectrum in Figure 9.7a. Its features at B through E could not account for the experimental spectrum by themselves, so contribution from different neutral radicals whose features coincided with those of the experimental... 

El-Agamey, A. and McGarvey, D.J. 2005. First direct observation of reversible oxygen addition to a carotenoid-derived carbon-centered neutral radical. Org. Lett. 18 3957-3960. 

Fresh peppers are excellent sources of vitamins A and C, as well as neutral and acidic phenolic compounds (Howard and others 2000). Levels of these can vary by genotype and maturity and are influenced by growing conditions and processing (Mejia and others 1988 Howard and others 1994 Lee and others 1995 Daood and others 1996 Simmone and others 1997 Osuna-Garcia and others 1998 Markus and others 1999 Howard and others 2000). Peppers have been reported to be rich in the provitamin A carotenoids (3-carotene, a-carotene, and (3-cryptoxanthin (Minguez-Mosquera and Hornero-Mendez 1994 Markus and others 1999), as well as xanthophylls (Davies and others 1970 Markus and others 1999). Bell peppers have been shown to exert low antioxidant activity (Al-Saikhan and others 1995 Cao and others 1996 Vinson and others 1998) or may even act as pro-oxidants (Gazzani and others 1998). 

There is no solid evidence that relates human aging and reduction of carotenoid absorption. In some studies, old people have shown a lower (3-carotene absorption than that of young people (Madani and others 1989), whereas the opposite has also been reported by other studies (Sugarman and others 1991). The absorption of lipid-soluble substances, including carotenoids, is affected by any disease related to the digestion and absorption of fats (West and Castenmiller 1998). Inadequate production of lipase and bile as well as an inadequate neutralization of the chyme in the duodenum affect carotenoid bioavailability (Guyton and Hall 2001). 

Finally, radical cations can be generated in solution by different types of pulse radiolysis225. Like PET, this is inherently a method for transient spectroscopic observations, but it has proved to be invaluable in investigations of dimer cations, e.g of polyenes, which form spontaneously upon diffusion of radical cations in the presence of an excess of the neutral parent compound, but a discussion of the electronic structure of such species is beyond the scope of this review. Pulse radiolysis is of interest in the present context because it allows the observation of large carotenoid radical cations which are difficult to create in solid-state or gas-phase experiments... 

Extracts were further purified on neutral alumina cartridges conditioned by passing through 5 ml of hexane. Extracts were loaded in hexane and washed by 5 ml of hexane. The at- and /1-carotenes were removed by 3.5 ml of acetone-hexane (10 90, v/v), other carotenoids were eluted with acetone-hexane 30 70 and 70 30 v/v. Prepurification of pigments was performed in subdued light under a stream of nitrogen. Analyses were carried out in a C30 column (250 X 4.6 mm i.d., particle size 5/tm) using isocratic mobile phase composed of methyl-ferf-butyl ether (MTBE)-methanol (3 97 and 38 62, v/v) at a flow rate of 1 ml/min. The column was not thermostated separations were achieved at room temperature (about 23°C). Carotenoids were detected at 453 and 460 nm (lutein). The... 

The elimination of arenes is not limited to the radical cations of the carotenoids. Just as the neutral compounds themselves also tend to undergo (thermal) cyclization followed by arene loss, the protonated analogues, e.g. ion 82 generated by Cl or fast atom bombardment (FAB) mass spectrometry are prone to eliminate one or even two arene molecules as well (Scheme 26)270. 

Radical cations may serve as acids by deprotonation and formation of the corresponding neutral radicals. This has been often shown in rigid matrices. An analogous reactivity was established in the radical cations of vitamin E type137 and carotenoids (see above)138. The latter lead to the formation of didehyd-rodiiners. 

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