CarotenoidsReferences:

Dewick, Chapter 5Hurst, 2nd edition, Chapter 6

Jake Stupalski, carotenoid chemist

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.

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.

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 carotenoid lycopene, found in tomato (Lycopersicon esculente)

• 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

Cyclizations lead to the variety of carotenoid structures

Carrots(Daucus carota)

Oxygenated carotenoids= xanthophylls(more common in fruits)

mango, persimmonmaizespinach

Capsicum(peppers)

brownalgae

Some common carotenoids with cyclic end structures:

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 (-carotene, -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.

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.)

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

OH

HO

Cranberries containlutein too!

Roles of carotenoids in nature:• Antioxidants, protection from radiation• Cleavage of -carotene by -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 unsubstituted -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)

Carotenoids provide color (attractants)• Carotenoids, together with chlorophylls and

anthocyanins, give most natural food colors• -carotene is most plentiful in green plant

tissues, followed by lutein & violaxanthin

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

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!

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

Eat your spinach salad with olive oil!

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

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

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

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

HPLC onC30 columnused isocraticelution with 85:152% NH4OAcin MeOH/MTBE