Carotenoids

1. Carotenoids References: Dewick, Chapter 5 Hurst, 2nd edition, Chapter 6 Jake Stupalski, carotenoid chemist

2. What are carotenoids? • Tetraterpenes (C40) • Long symmetrical chains of conjugated hydrocarbons usually with cyclization on either end. • Rings often have alcohol, epoxide, or carbonyl groups • Highly conjugated systems result in brightly colored yellow and orange compounds.

3. Biosynthesis: • Tail-to-tail coupling of 2 molecules of C20 geranylgeranyl diphosphate (GGPP) • Result: ---In plants and fungi (cis) Z-phytoene. ---In bacteria: (trans) E-phytoene. • Dehydrogenation leads to lycopene.

4. Biosynthesis: • The E (trans) form of Phytoene is produced by bacteria • The Z (cis) form of Phytoene is produced in plants and fungi. • Isomerization to E (trans) occurs during desaturation process, leading to straight chain carotenoidlycopene, found in tomato (Lycopersiconesculente) • Conjugation is extended by a sequence of desaturations removing pairs of H’s alternately from each side of triene system. • Central conjugated triene prevents the type of folding and multiple cyclizations seen with squalene

5. Cyclizations lead to the variety of carotenoid structures Carrots (Daucus carota) Oxygenated carotenoids = xanthophylls (more common in fruits) maize spinach mango, persimmon brown algae Capsicum (peppers)

6. Some common carotenoids with cyclic end structures:

7. Health Benefits: • Carotenoids possess high antioxidant capacity, with ability to absorb harmful UV light • Observed beneficial bioactivities may or may not be linked to the antioxidant properties • Higher total carotenoid (b-carotene, a-carotene, and lycopene intakes associated with lower abdominal fat mass • Higher lycopene intake related to lower serum triglyceride concentrations –Sluijs et al; Journal of Nutrition May 2009, 139 (5), 987-992.

8. Health Benefits: • High blood serum levels of Lutein correlate with a reduced risk for age-related macular degeneration (Breithaupt, et al; J. Agric. Food Chem. 2002; 50, 7175-7181 ) • Increased intake of carotenoids, particularly lycopene, associated with protection against loss of bone density at the lumbar spine in women and the hip in men (Sahni, et al; American Journal of Clinical Nutrition; Jan 2009; 89(1), 416-424) • Carotenoids can modify membrane structure and properties, affect human immune response, cell-cell signaling (Hurst, ed, “Methods of Analysis for Functional Foods and Nutraceuticals”, 2nd edition, CRC Press, 2008.)

9. Good for your eyes and your colon? • Lutein and zeaxanthin are present in the human eye (macula) and are thought to protect the retina from oxidative stress • Dietary intake is correlated with a lower incidence of macular degeneration, a common eye ailment in aging population • A 2008 Korean study found these carotenoids also block growth of colon cancer cells (Cha, et al, J. Agric. Food Chem) • Lutein and zeaxanthin extracted from algae induced apoptosis in colon tumor cells Cranberries contain lutein too!

10. Roles of carotenoids in nature: • Antioxidants, protection from radiation • Cleavage of b-carotene by b-carotene-15,15’-dioxygenase in the liver produces retinal (used in vision process) which is reduced to retinol (vitamin A1 below) • Any carotenoid with an unsubstitutedb-ring works • Carotenoids are absorbed more efficiently from cooked foods than fresh • Retinol and its derivatives are found only in animal products – these provide some of our dietary needs. Cod-liver oil and halibut-liver oil are rich sources • Many xanthophylls also absorbed by human body, though not the epoxyxanthophylls (e.g. neoxanthin)

11. Carotenoids provide color (attractants) • Carotenoids, together with chlorophylls and anthocyanins, give most natural food colors • b-carotene is most plentiful in green plant tissues, followed by lutein & violaxanthin

12. Carotenoids in nature, cont’d • Invertebrates often contain protein-associated carotenoids that give green, blue, purple, gray coloration • e.g. exoskeletons, eggs of crustaceans • role: protective coloring for camouflage • in plants, carotenoids act as light-harvesting pigments and protect against photo-damage by scavenging peroxyl and singlet oxygen • plastids contain chlorophyll-carotenoid-protein complexes (photosystems) to collect light energy for photosynthesis • with ripening, chloroplasts turn to chromoplasts, forming lipid-associated carotenoids (plastoglobules) with structure changes that result in color change

13. Around one in two million lobsters is blue. A research study conducted by Professor Ronald Christensen at the University of Connecticut discovered that a genetic defect causes a blue lobster to produce an excessive amount of protein.[5] The protein, and a red carotenoid molecule known as astaxanthin, combine to form a blue complex known as crustacyanin, giving the lobster its blue color.[6] Dennis Hoey (May 4, 2005). "Professor finds key to rare lobster color". MaineToday.com. http://travel.mainetoday.com/news/050404blue.shtml. Cooking releases the free carotenoid from the protein, turning the lobster red!

14. Carotenoid Esters: • Carotenoids in fruits and flowers may be esterified with fatty acids (e.g. lutein dipalmitate) • This occurs especially during ripening, altering solubility & polarity properties

15. Eat your spinach salad with olive oil!

16. Major nonpolar constituents of plants • Fatty acids and volatile or waxy esters • Triacylglycerols (fats & oils) • Phospholipids • Smaller terpenoids (monoterpene essential oils, sesqui and diterpenes) • Fat-soluble vitamins • Sterols and triterpenoids • Carotenoids

17. Separating nonpolar constituents of plants • Normal-phase chromatography on silica gel with gradient elution from nonpolar (hexane) to moderately polar (hexane/EtOAc) used to separate carotenoids and other large isoprenoids & lipids (next slide) • Fats & esters elute first, followed by carotenoid esters, free carotenoids and then sterols and triterpenoids • Compare to methods used for separation of polar phytochemicals – usually reverse-phase separation on C18 packing or a size-exclusion/polarity combination media like Sephadex LH-20

18. Sequential elution of cranberry carotenoids on silica gel with hexane/EtOAc gradient

19. Carotenoid analysis • TLC on silica gel or MgO/cellulose are commonly used with UV & PMA staining • Mobile phase varies with structure • Pet ether/acetone or hexane/EtOAc/EtOH, hexane/i-PrOH/MeOH (see table 6.9, Hurst) • Reverse-phase HPLC on C30 column Here, 90:10 hexane/ethyl acetate was used to analyze cranberry carotenoid fractions

20. HPLC on C30 column used isocratic elution with 85:15 2% NH4OAc in MeOH/ MTBE