Lutein in food supplements Irena Vovk 1, Breda Simonovska 1, Danica Agbaba 2 and Gordana Popović 2 1 National Institute of Chemistry, Ljubljana, Slovenia 2 University of Belgrade, Faculty of Pharmacy
Why lutein? Lutein and its structural isomer zeaxanthin were found as exclusive carotenoids in macula and retina of human eyes Essential lfor human vision i As strong antioxidants they might also have a beneficial effect on health, although the underlying mechanisms are mostly unknown and object of intensive investigations Higher intake of both micronutrients, resulting in higher plasma and tissues concentrations, was recommended for prevention of the age-related macular degeneration and cataract Numerous different formulations of food supplements containing lutein recently appeared on the market
Marigold Calendula officinalis L. (lutein about 10% of the total carotenoids) Tagetes erecta L. (lutein about 90% of the total carotenoids) China - the most important supplier of lutein from Tagetes erecta L.
Lutein and zeaxanthin in the diet Lutein (spinach, kale, orange, mango, egg yolk, E161b) Zeaxanthin (corn, E161h) Estimation of the average daily intake of lutein with the diet: 2 mg (usually less!) and about 10 times less zeaxanthin Recommended daily dose:? Toxicity: 20 mg is GRAS (Generally recognized as safe) f) High intakes can result in carotenodermia, a harmless and reversible hyperpigmentation p
Analytical problems Different contents per unit (e.g. 0.25 20 mg) Lutein present: as a mixture of lutein esters with saturated high fatty acids or free (not always declared) Difficult extraction of lutein or lutein esters from different formulations Stability Separation and identification
USP PHARMACOPEIA 2007, 2009 LUTEIN As purified fraction from saponified oleoresin of Tagetes erecta L. LUTEIN PREPARATION A combination of lutein with one or more inert substances in solid or liquid form PRINCIPLE A combination of spectrophotometric determination of total carotenoids and HPLC determination based on peak area % belonging gto all-trans-lutein
USP PHARMACOPEIA 2007, 2009 LIMITATIONS Only free all-trans-lutein (geometric isomers not even mentioned) Analysis of lutein esters impossible Other carotenoids interfere Yellow flavonoids interfere High consumption of organic solvents for a questionable extraction Error in volume definition for calculation of lutein content in solid lutein preparations without proteins Not useful for determination of lutein Not useful for determination of lutein in most of the food supplements.
Food supplements with lutein
Appearance of units (tablets, capsules) ) Tablet (lutein not evenly distributed in placebo) Tablet (evenly colored) Capsule with pastous content Capsule with oily content t Two-pieces capsule with dry content
Additional bioactive ingredients in food supplements with lutein Vitamin C (30-180 mg) Vitamin E (4-30 mg) Flavonoids (as bioflavonoids, extracts of blue berry, grape skins, pycnogenol etc) Zn (2.5-10 mg) Fish oil, soy oil Some preparations p contain additional bioactive compounds (mainly vitamins and minerals) Additional substances can be present as usual carriers in pharmaceuticals.
Development of analytical method for the determination of lutein PRINCIPLES Grinding and homogenization of samples which enable weighing of small aliquots Optimized saponification (concentration of base, solvent, time, temperature) Optimized extraction from basic solution Preparation of standard solutions for calibration and samples in the same way HPLC determination of all-trans-lutein, all-trans-zeaxanthin, cis-isomers of lutein
A 0,6 05 0,5 0,4 0,3 02 0,2 0,1 0 350 400 450 500 550 wavelength (nm) Published absorptivities iti enable determination of lutein concentration OH HO lutein OH HO zeaxanthin
60 40 20 Standard after saponification mau 13-cis-lutein 13'-cis-lutein all-trans-luteinn all-trans-zeaxanthin 9-cis-lutein 9'-cis-lutein 0 60 40 0 2 4 6 8 10 12 14 16 18 20 Minutes Sample M8 mau 13'-cis-lutein 13'-cis-lutein all-trans-lutein 9 9' all-trans-zeaxanthin -cis-lutein '-cis-lutein 20 0 0 2 4 6 8 10 12 14 16 18 20 Minutes
Validation of the developed analytical method PARAMETERS Selectivity Range Linearity System precision Intra-day and inter-days precision Accuracy LOD, LOQ Stability
Accuracy Spiking of placebo with free lutein or lutein esters at 3 levels, 3 replicates for each level Criteria: recovery 95-105%, RSD 5% Free lutein Lutein esters Level Mean recovery RSD (n=3) Mean recovery RSD (n=3) (%) (%) (%) (%) (%) 50 103.28 1.63 101.00 0.91 100 100.27 486 4.86 96.10 236 2.36 140 98.24 1.49 98.43 2.96
Additional characteristics of the developed method Usable for all kinds of samples (tablets, capsules with dry, pastous or oily content) t) Selective for lutein and zeaxanthin Determination of free lutein and zeaxanthin or a mixture of lutein esters Determination of 4 cis isomers of lutein Used organic solvents with low toxicity Low consumption of organic solvents Small mass of homogenised sample needed
lue of lut tein % of dec clared va 160 140 120 100 80 60 40 20 USP 2009 limits Results 0 1 2 3 4 5 6 7 8 9 101112131415161718192021222324252627282930 Food supplements One sample without declared content: 2.31 mg/unit
25 Results declared determined lutein per uni it (mg) 20 15 10 % of declare ed value of lutein 160 140 120 100 80 60 40 20 0 1 2 3 4 5 6 7 8 9 101112131415161718192021222324252627282930 Food supplements GRAS Average daily intake from food 5 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Food supplements
Conclusion The results of 31 analysed samples showed the importance of control of lutein in food supplements % of declared content of lutein Number of samples Without lutein 5 1-50 4 51-89 5 90-130 14 131-145 2 The consumer should consider the content of lutein in mg per unit (0.25 20 mg!) and expired date (stability!)
Grant from the: Acknowledgements Bilateral project (Slovenia-Serbia; BI- RS/08-09- 025): Determination of active ingredients in dietary supplements