Polar xanthophylls

However, complete hydrolysis of carotenoid esters sometimes is not achieved in 1 to 3 hr. The saponification degree can be verified easily by the presence of carotenol ester peaks eluting later than the peaks of P-carotene on reversed phase columns. Retinol palmitate, added as an internal standard to orange juice, also serves to indicate whether saponification is complete, since it is converted to retinol which elutes at lower retention time. The mixture is subsequently washed with water until free of alkali in a separatory funnel. Other more polar solvents such as CH2CI2 or EtOAc, and diethyl ether alone or mixtured with petroleum ether can be used to increase the recovery of polar xanthophylls from the water phase. 

The involvement of mitochondria in the pro-apoptotic effects of carotenoids has been clearly demonstrated by the fact that P-carotene induces the release of cytochrome c from mitochondria and alters the mitochondrial membrane potential (Aym) in different tumor cells (Palozza et al., 2003a). Moreover, the highly polar xanthophyll neoxanthin has been reported to induce apoptosis in colon cancer cells by a mechanism that involves its accumulation into the mitochondria and a consequent loss of mitochondrial transmembrane potential and releas of cytochrome c and apoptosis-inducing factor (Terasaki et al., 2007). 

Terasaki, M., Asai, A., Zhang, H. and Nagao, A. 2007. A highly polar xanthophyll of 9 -cis-neoxanthin induces apoptosis in HCT116 human colon cancer cells through mitochondrial dysfunction. Mol Cell Biochem 300 227-237. 

The limits of detection for carotenoids using FAB-MS and LSIMS are not as low as with most other ionization techniques (Schmitz et al., 1992). Therefore, >10 pmol of each carotenoid should be loaded onto the direct insertion probe per analysis. The matrix, 3-nitrobenzyl alcohol, has been effective in facilitating the ionization of all types of carotenoids. However, more polar matrices such as glycerol or thioglycerol might be useful for the FAB-MS or LSIMS analysis of polar xanthophylls such as astaxanthin. Because glycerol and thioglycerol are poor solvents for hydrophobic compounds, they are unlikely to solvate and thus facilitate the ionization of the nonpolar carotenes such as (3-carotene. 

Natural product samples typically contain both the nraipolar carotenes and the more polar xanthophylls. Whatever the method used, the chromatographic process should be able to cope with this polarity range. 

Ruban AV, Horton P, and Young AJ. 1993. Aggregation of higher-plant xanthophylls—Differences in absorption-spectra and in the dependency on solvent polarity. Journal of Photochemistry and Photobiology B-Biology 21(2-3) 229-234. 

Solvents with different polarities and refractive indexes significantly affect carotenoid optical properties. Because the refractive index is proportional to the ability of a solvent molecule to interact with the electric held of the solute, it can dramatically affect the excited state energy and hence the absorption maxima positions (Bayliss, 1950). Figure 7.2a shows three absorption spectra of the same xanthophyll, lutein, dissolved in isopropanol, pyridine, and carbon disulfide. The solvent refractive indexes in this case were 1.38, 1.42, and 1.63 for the three mentioned solvents, respectively. 

Carotenoids were discovered during the nineteenth century. Wachen in 1831 proposed the term carotene for the hydrocarbon pigment crystallized from carrot roots Berzelius called the more polar yellow pigments extracted from autumn leaves xanthophylls and Tswett separated many pigments by column chromatography and called the whole group carotenoids. ... 

Chemical properties of carotenoids play an important role in carotenoid micellarization and, therefore, bioavailability. Apolar carotenoids (carotenes) are generally incorporated in the central region, which is highly hydrophobic, of the oil droplets, whereas polar carotenoids (xanthophylls) are localized on the surface, and therefore xanthophylls are more easily micellarized and absorbed than carotenes (Borel and others 1996). van het Hof and others (2000) found in humans that lutein is five times more bioavailable than (3-carotene. 

Free xanthophylls, both endogenous and present in the saponified samples, are more polar and extract less efficiently into lipophilic solvents. Frequently, the addition of a polar organic solvent (tetrahydrofuran, methylene chloride, diethyl ether) is required to thoroughly extract them from the sample matrix and aqueous phase. 

In normal-phase chromatography, polar components are more strongly retained than nonpolar components. Thus, hydrocarbon carotenes elute quickly while xanthophylls are retained and separated. This approach provides a more complete separation of polar carotenoids and their geometric isomers. This protocol is useful to the analyst that is specifically interested in the xanthophyll fraction of a sample. 

Electrospray ionization will produce molecular ions, M+, with almost no fragmentation for carotenes and many xanthophylls. As the polarity of the carotenoid increases, the prob-... 

Water-containing plant materials need to be extracted with polar solvents such as acetone, methanol, or ethanol that can take up water. Freeze-dried plant tissues and freeze-dried juices can be directly extracted with diethyl ether, which contains traces of water and is more polar than light petrol or hexane. Pure light petrol or hexane are less suitable, because more polar pigments, such as Chi b or xantho-phylls, are only partially extracted from freeze-dried plant samples. A few drops of acetone or ethanol added to light petrol or hexane will, however, guarantee a complete extraction. This mixture will extract Chi a, Chi b, and all carotenoids—including xanthophyll esters and secondary carotenoids that are present in many fruits and juices—from the freeze-dried plant material. 

While esterified xanthophylls can be extracted using solvent mixtures similar to those used for hydrocarbon carotenes, nonesterified xanthophylls are generally extracted using more polar solvents. For example, xanthophylls have been extracted from spinach using a mixture of methanol and tetrahydroftiran (Kopas-Lane and Warthesen, 1995). Acetone alone has also been used to extract lutein and zeaxanthin from a variety of fresh and processed vegetables (Updike and Schwartz, 2003). However, for the extraction of a wide range of carotenoids, a mixture of polar and nonpolar solvents works best. 

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