Lutein separation

A further thirty years were to pass before Kuhn and his co-workers (3) successfully repeated Tswetf s original work and separated lutein and xanthine from a plant extract. Nevertheless, despite the success of Kuhn et al and the validation of Tswett s experiments, the new technique attracted little interest and progress continued to be slow and desultory. In 1941 Martin and Synge (4) introduced liquid-liquid chromatography by supporting the stationary phase, in this case water, on silica in the form of a packed bed and used it to separate some acetyl amino acids. 

Kostic, D., White, W.S., and Olson, J.A., Intestinal absorption, serum clearance, and interactions between lutein and 3-carotene when administered to human adults in separate or combined oral doses. Am. J. Clin. Nutr, 62, 604, 1995. 

Separation of lutein esters from complex plant extract mixture... 

The most common mobile phase is a gradient of petroleum ether or hexane with increasing concentrations of acetone or diethyl ether. Development of the column should be optimized for each sample to afford a quick and effective separation to avoid band broadening. The separation can be followed visually. The most non-polar a- and 3-carotenes are eluted first as a yellow band followed by the chlorophylls and other more polar carotenoids like cryptoxanthin, lutein, and zeaxanthin that frequently fuse together and appear as a single band. ... 

Figure 6.2.2 shows the separations of mixtures of standards on a monomeric C,g column and also on a polymeric C30 column. The elution order on the monomeric C18 column is, as expected, first the dihydroxy xanthophyUs (lutein and zeaxanthin), followed by the monohydroxy compounds (rubixanthin and P-cryptoxanthin), and finally by the carotenes (y-, a-, and p-carotene). However, on the C30 column, rubixanthin and y-carotene, both with 1 acyclic /-end group, eluted after a- and P-carotene, with two cyclic end groups. 

Dachtler, M. et al.. Combined HPLC-MS and HPLC-NMR on-line coupling for the separation and determination of lutein and zeaxanthin stereoisomers in spinach and in retina. Anal. Chem., 73, 667, 2001. 

As has been pointed out earlier in this chapter, the dietary consumption and historical medicinal use of carotenoids has been well documented. In the modern age, in addition to crocin, 3.7, and norbixin, 3.8, several carotenoids have become extremely important commercially. These include, in particular, astaxanthin, 3.6 (fish, swine, and poultry feed, and recently human nutritional supplements) lutein, 3.4, and zeaxanthin, 3.3 (animal feed and poultry egg production, human nutritional supplements) and lycopene, 3.2 (human nutritional supplements). The inherent lipophilicity of these compounds has limited their potential applications as hydrophilic additives without significant formulation efforts in the diet, the lipid content of the meal increases the absorption of these nutrients, however, parenteral administration to potentially effective therapeutic levels requires separate formulation that is sometimes ineffective or toxic (Lockwood et al. 2003). 

FIGURE 4.4 Separation of lutein stereoisomers, comparison between C18 and C30 phases. (From Dachtler, M. et al., J. Chromatogr. B, 211, 1998. With permission.)... 

Figure 4.7 shows the structures of important carotenoids (all-E) lutein, (all-E) zeaxanthin, (all-E) canthaxanthin, (all-E) p-carotene, and (all-E) lycopene. Employing a self-packed C30 capillary column, the carotenoids can be separated with a solvent gradient of acetone water=80 20 (v/v) to 99 1 (v/v) and a flow rate of 5 pL min, as shown in Figure 4.8 (Putzbach et al. 2005). The more polar carotenoids (all-E) lutein, (all-E) zeaxanthin, and (all-E) canthaxanthin elute first followed by the less polar (all-E) p-carotene and the nonpolar (all-E) lycopene. Figure 4.9 shows the stopped-flow II NMR spectra of these five carotenoids. The chromatographic run was stopped when the peak maximum of the compound of interest reached the NMR probe detection volume.

Recently, due to increased interest in membrane raft domains, extensive attention has been paid to the cholesterol-dependent liquid-ordered phase in the membrane (Subczynski and Kusumi 2003). The pulse EPR spin-labeling DOT method detected two coexisting phases in the DMPC/cholesterol membranes the liquid-ordered and the liquid-disordered domains above the phase-transition temperature (Subczynski et al. 2007b). However, using the same method for DMPC/lutein (zeaxanthin) membranes, only the liquid-ordered-like phase was detected above the phase-transition temperature (Widomska, Wisniewska, and Subczynski, unpublished data). No significant differences were found in the effects of lutein and zeaxanthin on the lateral organization of lipid bilayer membranes. We can conclude that lutein and zeaxanthin—macular xanthophylls that parallel cholesterol in its function as a regulator of both membrane fluidity and hydrophobicity—cannot parallel the ability of cholesterol to induce liquid-ordered-disordered phase separation. 

The presence of only two dietary carotenoids in the retina, lutein and zeaxanthin, out of about 14 normally present in the plasma indicates their highly specific uptake and retention (Bernstein et al., 2001 Bone and Landrum, 1992 Bone et al., 1988,1997,1993 Davies and Morland, 2004 Khachik et al., 1997, 2002). The retina-blood barrier is formed by the tight zonulae occludentes of the endothelial cells in the inner retina and of the RPE, a monolayer of cells which separates the outer retina from its choroidal blood supply (Strauss, 2005). 

Several researchers have tried to isolate cellular CBPs from the silkworm. In Nakajima s study (1963), the whole midgut mucosa was homogenized and the proteins separated with a gel-filtration chromatography column. Carotenoids were found in certain fractions containing proteins, suggesting the existence of CBPs in the midgut. Jouni and Wells purified a 35 kDa protein containing lutein... 

Tissue samples obtained from the different colored regions of the larvae were separately analyzed by HPLC. The white, black, and yellow bands of Monarchs all contained a single, major carotenoid component, lutein (all / -3,3 - d i h yd roxy-13, e - ca ro t e n e), Figure 25.3a. The amount of lutein present in the black and white bands was markedly lower ( 15x) than that in the yellow bands, see below. A small quantity of 13-m-lulein and zeaxanthin were observed to elute immediately following lutein in the chromatogram and the lutein peak was preceded by a unique metabolite that is formed by the cleavage of lutein, see Section 25.4. 

FIGURE 25.7 Analysis of sites within a single band for four separate animals shows a clear gradient in the concentration of lutein from head to tail. The average ratio of head position to middle position (A/B) is 1.9 0.6 and that of the head to tail position (A/C) is 2.8 0.9. 

The thyreotropic hormone166 from pituitary extracts appeared to be an entity separate from the luteinizing hormone and contained nitrogen, 13% carbohydrate, 3.5% and D-glucosamine, 2.5%. 

Another study employed both TLC and HPLC for the analysis of carotenoids of Calendula officinalis L. TLC separation of all E(trans)-a,3-carotene, cryptoxanthin, zeaxanthin and lutein was performed on a silica layer using petroleum ether-j-propanol-CIICI, (90 10 70 v/v). The same carotenoid pigments were separated by RP-HPLC using an ODS column (250X4 mm, i.d.). The organic modifiers were methanol, THF and ethyl octane. The flow rate was 1 ml/min, pigments were detected at 440 nm [20],... 

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... 

Fig. 2.26. Reversed-phase HPLC separation of (a) Sobrasada sausage extract and (b) saponified Sobrasade sausage extact in an ODS column at maximum absorbances at each point in time. Peak identification 1 - 2, 4 - 6, 8, 12, 14-17 = unidentified free 3 = capsorubin 7 = violaxanthin 9 = capsanthin 10 = anteraxanthin 11 = cw-capsanthin 13 = lutein and zeaxanthin 18 = cantaxanthin, internal standard 19 = cryptoxanthin 20, 24, 25, 28 = unidentified monoester 21 = /J-cryptoxanthin 22 = capsorubin monoester 23, 26, 27, 29 = capsanthin monoester 30, 31 = lutein-zeaxanthin monoester 32 = /1-carotene 33 = cis-f)-carotene 34, 37, 39, 41, 43 = capsanthin diester 35 = capsorubin diester 36, 38, 40, 42, 44 = unidentified diester. Reprinted with permission from J. Oliver et al. [56],...

---