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Nutrition for Optimal Wellness
Plant Xanthophylls in Eye Health and Their Potency Determination in Nutraceuticals
By Ted Waszkuc, Ph.D., NOW R&D and Methods Development
“Lutein Esters” (product code # 3058) is the NOW Foods product containing natural carotenoids extracted from marigold (Tagetes erecta L) flower petals, known under the trade name of Xangold®. Its main components are the carotenols lutein and zeaxanthin occurring in their natural form of esters with fatty acids. Since a variety of acids can be connected with one or two hydroxyl groups in carotenols, the extract consists of several (usually more than 10) structurally different esters.1 It presents a challenge from an analytical viewpoint.
Carotenoids are widespread plant pigments accumulated in leaves and flowers (marigold, daffodils, tulips), also found in fruits and vegetables that are the major sources of these micronutrients for humans. Chemically they are tetraterpenoid compounds with at least 10 conjugated double bonds. This extended conjugation explains their yellow or orange/red color and extreme tendency to become oxidized. For that reason, carotenoids are potent antioxidants acting as efficient singlet oxygen and peroxy radical scavengers.2
Carotenols, better known under the traditional name of xanthophylls, are a subclass of carotenoid compounds, carrying one or two hydroxyl groups in their structures. Found in the leaves of most plants, xanthophylls function in photosynthesis along with chlorophyll as accessory light-harvesting pigments. They also serve as protectants for plants against photo-oxidative damage by quenching harmful oxygen species. Xanthophylls predominate in autumn leaves, where they are degraded less rapidly then the green chlorophyll pigment. Animals can not produce them, but they do take these nutrients in from foods. The yellow color of chicken egg yolk 3 and the pink color of salmon flesh,4 for example, are produced in this way.
The major xanthophylls are lutein, zeaxanthin, and to a much lesser extent cryptoxanthin. They may be esterified with saturated or unsaturated fatty acids and have been detected in spinach,5 corn,6 peppers, apricots, peaches, pink grapefruit, oranges, and other fruits and vegetables popular in the human diet.
The xanthophylls lutein and zeaxanthin, so-called ‘retinal carotenoids’, have been found to accumulate in the central region of the eye retina, where the photoreceptors reach their maximum density, giving the eye its highest visual acuity. The yellow pigmentation has given this region the Latin name macula lutea, or yellow spot. Xanthophylls can protect the vulnerable lipid-rich outer membrane of photoreceptors against photo-oxidative damage, especially by absorbing short-wave blue light and deactivating reactive oxygen species often generated within the retina. Increased intake of lutein/zeaxanthin rich foods8 like kale, spinach and corn - or lutein and zeaxanthin supplementation - showed an increase in macular pigmentation, indicating a beneficial role of these carotenoids in eye health.9     
High performance liquid chromatography (HPLC) is the method of choice in quantitative analysis of natural constituents derived from plant material. The technique relies on separation of components present in an analyzed sample. The HPLC instrument records the presence of each component in the mixture as a peak on the chromatogram (a graph), with the peak size related to the amount of a given component in the sample. In order to produce accurate and reliable results the HPLC test requires the use of highly purified compounds - called reference standards - with exactly the same chemical structure as components present in the extract analyzed.
Most of the carotenol esters we deal with in marigold extract are not commercially available as pure chemicals and so can not be measured directly using HPLC and reference standards. Chemically, esters are composed of alcohol (here ‘carotenol’) and an acid (here ‘fatty acid’). To proceed with chromatographic analysis of lutein esters the sample must be worked out using specific chemical processes to break down esters to their parent compounds carotenols (a purer form that does have reference standards) and fatty acids. After de-esterification in mild alkaline conditions,10 lutein, zeaxanthin and cryptoxanthin potency can be accurately measured in our lab. This assures that NOW® Lutein Esters meets our label claims for identity and potency.
1  Rivas, J.D., Reversed-phase high-performance liquid chromatographic separation of lutein and lutein fatty acid esters from marigold flower petal powder. Chromatography, 137, 443-447 (1987)
2  Bruneton, J., Carotenoids in Pharmacognosy, Phytochemistry Medicinal Plants, Lavoisier Publishing Inc. (1999) p. 769
3  The Merck Index, thirteenth ed. 10120, (2001)
4  Domininguez, A., et al., Delivery of astaxanthin from Haematococcus pluvialis to the aquaculture food chain.  Aquaculture 250, 424-430 (2005)
5  Aman, R., et al., Application of HPLC coupled with DAD, ApcI and NMR to the analysis of lutein and zeaxanthin stereoisomers in thermally processed vegetables  Food Chemistry 92, 753-763 (2005)
6  Scott, C.E., et al., Comparison of carotenoid content in fresh, frozen and canned corn.  J. Food Comp. Anal. 18, 551-559 (2005)
7  Wingerath, T., et al., Xanthophyll esters in human skin.  Archives of Biochem. Biophys. 355, 271-274 (1998)
8  Curran-Celentano, J., et al., Relation between dietary intake, serum concentrations, and retinal concentrations of lutein and zeaxanthin in adults in a Midwest. Am.J.Clin.Nutr. 74, 796-802 (2001)
9  Landrum, J.T., et al., A one year study of the macular pigment: the effect of 140 days of a lutein supplement.  Exp.Eye Res. 65, 57-62 (1997)
10  Minguez Mosquera, M.I., et al., Study of lability and kinetics of main carotenoid pigments of red pepper in the de-esterification reaction.  J.Agric. Food Chem. 46, 566-569 (1998)