1. 1

2. The Color Phenomenon Color is a perception that is manifested in response to a narrow span of the electromagnetic spectrum emitted by light sources (e.g., sunlight). Light itself has no color and color does not exist by itself; it only exists in the mind of the viewer What is light? A vibration in an electromagnetic field through which energy is transported. Since it is an electromagnetic field, it does not require a medium. 2

3. The Electromagnetic Spectrum Most wavelengths of light can not be seen by the human eye.  The visible part of the electromagnetic spectrum lies between ultraviolet and infrared light (between about 400 and 700 nm).  The higher the frequency (shorter the wavelength), the higher the photon energy. Radio waves are at the long wavelength end of the spectrum and gamma rays are at the short wavelength end of the spectrum.  The visible part of the electromagnetic spectrum lies between ultraviolet and infrared light (between about 400 and 700 nm).  The higher the frequency (shorter the wavelength), the higher the photon energy. Radio waves are at the long wavelength end of the spectrum and gamma rays are at the short wavelength end of the spectrum. 3

4. 4

5.  Pigments are compounds that absorb light in the wavelength range of the visible region. This absorption is due to a molecule-specific structure (chromophore) that captures the energy from a radiant source.  Some energy is not absorbed and is reflected and/or refracted; this energy is captured by the eye and generates neural impulses, which are transmitted to the brain, where they could be interpreted as a color. 5

6. The Dry Color Manufacturers Association makes a clear distinction between pigment and dyes: 1. Pigment is a colored, black, white, or fluorescent particulate organic or inorganic solid, which is usually insoluble and, essentially, physically and chemically unaffected by the vehicle or substrate into which it is incorporated. Thus, the pigmentation effect is by selective absorption and/or by scattering of light; a pigment will retain its crystalline or particulate structure. 2. Dyes are soluble in the carrying medium and therefore crystalline/particulate features are lost in solution when a dyestuff is used to impart color to a material. 6

7. In the latter definition, the difference between pigment and dye is emphasized. However, other authors prefer to use the more generic term colorant Colorants are defined as substances that modify the perceived color of objects, or impart color to otherwise colorless objects.  With this definition, pigments and dyes are grouped within the term colorants. It is reasoned that if only solubility is considered, the same substance could be a dye or a pigment depending on how it is used.  It is important to be aware of such differences, but in our discussions we will use the terms colorants and pigments as synonymous. 7

8.  Chlorophylls and carotenoids are the most abundant pigments in nature. They are involved in fundamental processes, and life on Earth depends on them.  Chlorophyll is not found in animals but carotenoids accumulate in some organs (e.g., eyes) and tissues (e.g., skin of fish, bird plumage).  In general, animal carotenoids are obtained from the common diet. Other pigments are also found in animals, some have important functions (e.g.,heme proteins, riboflavin), whereas the function of others is not yet completely clear (e.g., melanins, flavonoids). 8

9. 1. Tetrapyrroles: Chlorophylls (Green), Bilins 2. Isoprenoid derivatives:  Carotenes (e.g., β-carotene, lycopene) Yellow-red  Xanthophylls (e.g., lutein, zeaxanthin) Yellow 3. N-Heterocyclic compounds  Betalains Yellow-red 4. Benzopyran derivatives:  Flavonoids (e.g., anthocyanins, flavonols,flavones, anthochlors), Blue-red, yellow-white, white, cream, yellow 5. Quinones:Anthraquinone (e.g., carminic acid) Red-purple 9

10.  Tetrapyrroles are very important in living organisms and probably every organism has the ability to synthesize them.  These pigments can be either linear or cyclic structures, but the pyrrole ring is common in both kinds of molecules  In the tetrapyrrole group, the chlorophyll subgroup is the most important; this subgroup is mainly present in the chloroplasts of higher plants and most algae 10

11.  Chlorophylls - usually most abundant and most important pigments in light harvesting  located in protein complexes in internal membranes of chloroplasts  Contain tetrapyrrole ring (chlorin) similar to heme, but contains Mg 2+  Chlorophylls a (Chl a) and b (Chl b) in plants 11

12. Leaves contain millions of chloroplasts Leaf cross-section Cells containing chloroplasts Chlorophyll is found and Photosynthesis takes place in the chloroplasts of plant cells 12

13. chlorophyll a – primary pigment in plants and cyanobacteria -absorbs violet-blue and red light chlorophyll b – secondary pigment absorbing light wavelengths that chlorophyll a does not absorb 13

14. 14

15. H CH 2 CH 3 H CH 3 CO 2 CH 3 O H H 3 C H 3 C N NN N CHH 2 C M g R CH 2 CO 2 CH 3 CH 3 CH 3 CH 3 Chlorophyll 15

16. 16

17. 17

18. Photosynthesis Light Dependent Reaction – Energy from the high energy electron of chlorophyll is used to make ATP and NADPH Light Independent Reaction (Calvin Cycle) – ATP and NADPH is used to make glucose from CO 2 18

19. 19

20. 20

21. 21

22. 22 1. Tetrapyrroles: Chlorophylls (Green), Bilins 2. Isoprenoid derivatives:  Carotenes (e.g., β-carotene, lycopene) Yellow-red  Xanthophylls (e.g., lutein, zeaxanthin) Yellow 3. N-Heterocyclic compounds  Betalains Yellow-red 4. Benzopyran derivatives:  Flavonoids (e.g., anthocyanins, flavonols,flavones, anthochlors), Blue-red, yellow-white, white, cream, yellow 5. Quinones:Anthraquinone (e.g., carminic acid) Red-purple 22

23.  Isoprenoids are also known as terpenoids and are widely distributed in nature. They are found in all the kingdoms where they develop multiple functions (hormones, pigments, phytoalexins).  The isoprenoid group has three main subgroups: quinones, carotenoids, and iridoids are the most important pigments in the isoprenoid group 23 2. Isoprenoid derivetives

24.  Carotenoids are compounds constituted by eight isoprenoid units (ip). The ip units are joined in a head- to-tail pattern, but the order is inverted at the molecule center  Lycopene is considered the first colored carotenoid in the biosynthesis of many other natural carotenoids and it is linear. 24 Carotenoids

25.  Carotenoids are pigments (C40) that naturally occur in chloroplasts and other photosynthetic organisms and absorb light for photosynthesis.  There are over 600 carotenoids including beta-carotene.  Carotenoids have been found to have antioxidative properties, reducing the risk of mortality from chronic illnesses.  Animals are incapable of producing carotenoids and must obtain them from their diet. E.g the pink in flamingos & red in lobsters are from carotenoids in their diets.  Carotenoids with only hydrogen and carbon in their molecules are collectively called carotenes; oxygenated derivatives are called xanthophylls. 25

26. CAROTENOIDS IMPORTANT PHYSICAL AND CHEMICAL PROPERTIES OF CAROTENOIDS Bind to hydrophobic surfaces Easily isomerized And oxidized Absorb light Scavenge or interact with free radicals Quench singlet oxygen Lipophilic 26

27. PRINCIPAL CAROTENOIDS IN FOODS  -Carotene  -Cryptoxanthin OH Lycopene Lutein HO OH 27

28. CAROTENOIDS HEALTH-PROMOTING FUNCTIONS AND ACTIONS ATTRIBUTED TO CAROTENOIDS Reduction of the risk of cataract formation Reduction of the risk of macular degeneration Provitamin A activity Immuno- enhancement Reduction of the risk of cancer Reduction of the risk of cardiovascular disease 28

29. Beta-carotene & Lycopene Most commonly known as the pigment that makes carrots orange, beta-carotene is a precursor of Vitamin A. Vitamin A, important for vision and as an antioxidant, is made from beta-carotene via beta-carotene dioxygenase. Lycopene is an intermediate in the production of beta- carotene, and is a bright red carotenoid. Lycopene is found in tomatoes, pink grapefruit, red bell peppers, etc., and has been found to have antioxidant activity. 29

30. 30 1. Tetrapyrroles: Chlorophylls (Green), Bilins 2. Isoprenoid derivatives:  Carotenes (e.g., β-carotene, lycopene) Yellow-red  Xanthophylls (e.g., lutein, zeaxanthin) Yellow 3. N-Heterocyclic compounds  Betalains Yellow-red 4. Benzopyran derivatives:  Flavonoids (e.g., anthocyanins, flavonols,flavones, anthochlors), Blue-red, yellow-white, white, cream, yellow 5. Quinones:Anthraquinone (e.g., carminic acid) Red-purple 30

31. Flavonoids (e.g., anthocyanins, flavonols, flavones, anthochlors), Blue-red, yellow-white, white, cream, yellow 31 3. Benzopyran derivatives:

32.  Flavonoids and anthocyanins are conspicuous plant pigments in nature that are responsible for the beauty and splendor of flowers, fruits, fruit tree blossoms and of the autumn leaves.  Flavones are responsible for the yellow and orange colors; and the anthocyanins are the source of red, violet and blue colors. These compounds occur mainly in higher plants and are less common in the lower orders. You don’t find them in algae, fungi or bacteria. 32

33.  The flavonoids play a major role in attracting insects to feed and pollinate these plants. Some of them also have a bitter taste and repel harmful insects like caterpillars.  Flavonoids are thought to be antioxidants, and play a major role in our diet, preventing the ravages of aging caused by free-radicals.  These compound have their biosynthetic origin in both the skimic acid pathway and the acetogenin pathway - they are of hybrid origin. 33

34. Anthocyanin Flower Pigments 34

35. Largest class of polyphenols: ~ 4,000 kinds Often responsible for yellow or red/blue/purple colors in flowers, fruits, vegetables Serve as antioxidants for the plants during photosynthesis Includes antioxidant, anti-inflammatory, cancer- preventative, & cardioprotective compounds 35

36. 36

37. I. Carotenoids as food colorants  Humans have utilized carotenoids as food colors for centuries: saffron, pepper, leaves, and red palm oil are some of the most used pigments.  These products comprise mixtures of pigments and other frequently unidentified substances; natural sources of carotenoids have persisted through the years and new ones have been introduced.  As previously mentioned, carotenoids have very important biological activities and their use as food and feed is common today and recommended largely due to their vitamin A and antioxidant activities, very important for the maintenance of body health. 37

38. 1. Carotenes  An oleoresin is obtained by solvent extraction of carrots or alfalfa grass and vegetable oil with subsequent removal of solvents.  The oleoresin contains oils, fats, and waxes of natural occurrence in the source material.  The permitted solvents for the extraction process are acetone, methanol, ethanol, propan-2-ol, hexane, and dichloromethane; their use is allowed in different food products.  The FDA has permitted the use of these sources of carotenes for coloring foods in general. 38

39. 2. Marigold  Marigold (Tagetes erecta L.) is an annual herb and its petals have a high content of carotenoids with values higher than 8 g of carotenoids/kg of dried petals. It is also interesting that only one carotenoid predominates in its composition — lutein — with more than 80%.  The main marigold products are flower meals and oleoresin, which is obtained by hexane extraction.  In the oleoresin, lutein is as ester with palmitate, myristate, and stearate in all the possible combinations.  It is used in feed pigmentation and in the European Union as a food additive. Marigold pigment can be further purified and mixed with a suitable vegetable oil, calcium silicate, and gelatin, among others, to obtain a product of food grade. This product is suitable for coloring pastas, vegetable oils, dairy and bakery products, as well as juices and mustard, among others. 39

40. 3. Paprika  Is obtained from the dried pods of sweet pepper (Capsicum annum).  To obtain paprika, pods are ground to produce a powder of deep red color and pungent flavor.  Paprika carotenoids are mainly capsanthin and capsorubin, which occur primarily as lauric acid esters.  The quality of paprika is commonly specified as color strength by measuring the absorbance at 460 nm in an acetone extract, as suggested by the American Spice Trade Association (ASTA).  Paprika oleoresin is an orangered oil-soluble extract that is obtained by using hexane as solvent. The use of paprika, as a spice, is limited to suitable savory products.The use of paprika powder and oleoresin is permitted by the FDA for coloring foods in general. 40

41. 4. Saffron  (Crocus sativus L.) is the most expensive spice used in the food industry and its consumption has shown increments because of its health beneficial properties.  This plant is native to Greece and Asia, and today is cultivated in various parts of the world. Iran and India are the major world suppliers of saffron, but its quality is not as high as that of Spain. 41

42.  The coloring power is mainly attributed to water- soluble carotenoids, the crocins, which are glycosyl esters of 8,8′-diapocarotene-8,8′-dioic-acid (crocetin).  The high solubility of saffron pigments in water is a strong advantage, compared to other carotenoids that require special formulations to be soluble or dispersible in aqueous food systems, and is the reason for their great application as a food colorant.  Saffron is basically used to impart a pure yellow color to rice and other foods; it is also employed as a spice.  Color is the major parameter for quality grading of saffron Its use as a coloring agent for food in general was established in 1966 by the FDA 42

43. Tomato (Lycopersicon esculentum) has a high carotenoid content with lycopene the main compound (80 to 90% of total carotenoids) followed by β- carotene. New tomato varieties with high and improved content have been developed; efforts have focused on developing tomatoes with higher β- carotene content, which are thus better sources of vitamin A. Lycopene products have begun to be commercialized.  However, it is clear that other modifications are required to use tomato extract as colorant because of its strong flavor. 43

44.  In the United States, extracts of grape are the only anthocyanin source approved by the FDA as a food colorant.  The commercial preparations are enocyanin and lees (sediment of grape juice tanks). The grape color extract is approved to be used in nonbeverage foods, whereas grape skin extract (enocyanin) is permitted in beverages. 44

45.  legislation, grape commercial extracts are used mainly for beverages, soft drinks, as well as confectionery products  In addition, the anthocyanins of red radish have good stability; maraschino cherries colored with radish anthocyanins have a shelf life of at least 6 months at 25°C, and their stability has been associated with the presence of acylated pelargonidin derivatives.  The proposed method to produce radish anthocyanins uses an abrasive peeler that is followed by pressing of the epidermal pulp; this strategy produces recoveries greater than 90%. 45

46. 46 Natural Colorants as Nutraceuticals

47. I. Carotenoids  Pathological processes, including cancer and strokes, in living organisms are commonly associated with an oxidative stress condition. Thus, antioxidant compounds are usually considered beneficial agents for prevention or treatment  As mentioned before, some carotenoids have a considerable high antioxidant activity and consequently a positive effect in human health  Carotenoids are membranal pigments: carotenes are apolar and immersed in membranes, showing little mobility, whereas xanthophylls are polar and have a variable position and mobility in membranes. 47

48.  As can be deduced, carotenes have good antioxidant activity against radicals generated inside the membrane.  On the other hand, the xanthophyll zeaxanthin has its hydroxyl groups exposed to the aqueous cellular media and it is able to react with radicals of that zone. The relative mobility of carotenoids has been associated with the fluidity of membranes; particularly, carotenoids have shown an effect on membrane permeability to oxygen and other substrates 48

49. Type of Carotenoid Biological Function 1. All : Effect in the immune response and in the intercellular communication, treatment of photosensitivity diseases  The use of algae (especially Phaeophyta) carotenoids diminishes the risks of being affected by certain types of cancer 2. β-Carotene: Treatment of certain kinds of cancer (e.g., smoking-related cervical intraepithelial neoplasia and cervical and stomach cancer), affects the immune response in rats and by this mean tumor growth is inhibited, inhibits lipid peroxidation, suppresses the increase of hormones related to stress syndrome. Suppresses the increase of hormones related to stress syndrome 49

50. 3. lycopene:  Has been evaluated in rats in relation to its role in colon carcinogenesis using azoxymethane as a chemical carcinogen.  Lycopene has been provided in the form of a 6% oleoresin, which is well absorbed and produces a reduction in the serum of the thiobarbituric acid reactive substances (TBARS), suggesting an important role in the protection against oxidative stress.  Moreover, the number and size of aberrant crypt foci (ACF) is reduced; thus, it could have a positive role against colon carcinogenesis. 50

51.  Lycopene is accumulated in tissues such as prostate, adrenals, and testes; consequently, a selective uptake of the carotenoid may be involved in a tissue-specific anticarcinogenic mechanism.  Also, it has been reported that tissues high in LDL receptors selectively accumulate lycopene, and a protective effect of this carotenoid is evident at a dose of 10 ppm, which is equivalent to two servings of tomatoes or tomato products per day. 51

52.  The anthocyanins of Hibiscus sabdariffa L. (Malvaceae) have been used effectively in folk medicines against hypertension, pyrexia, and liver disorders.  tert- Butylhydroperoxide (t-BHP) can be metabolized into free radical intermediates by cytochrome P-450 (in hepatocytes) or hemoglobin (in erythrocytes), which can subsequently initiate lipid peroxidation, affect cell integrity, and form covalent bonds with cellular molecules resulting in cell injury.  t-BHP causes leakage of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) and formation of malondialdehyde  (MDA) in hepatocyte cultures. It also mediates DNA damage in mammalian cells. These phenomena are similar to the oxidative stress occurring in the cell or tissue 52

53.  Oxidative stress is considered to play a prominent role in the causation of many conditions, for example, inflammation, aging, and cancer.  It is observed that Hibiscus anthocyanins inhibit lethal injury induced by t-BHP in rat primary hepatocytes and rat livers that might involve their ability to quench free radicals.  Hibiscus anthocyanins are able to quench the 1,1-diphenyl-2- picryhydrazyl (DPPH) free radicals in a dose-dependent manner; at 0.20 mg/ml they quench about 50% of the free radicals. It has also been pointed out that up to 0.50 mg/ml concentration they exhibit no toxic effect on the primary culture of hepatocytes. 53

54. .  Thus, Hibiscus pigments exhibit an antihepatoxicity effect against t- BHP-induced cytotoxicity, probably via their ability to quench free radicals and decrease MDA formation. It is suggested that consuming soft drinks containing Hibiscus pigments is sufficient to reach the lowest dose (50 mg/kg) that showed some antioxidant activity in this study.  Therefore, we suggest that daily consumption of Hibiscus anthocyanins might be effective in lowering oxidative damage in living systems. 54

55. Used in Sources ColorsSubstance candy, fruit beverages, ice cream, yoghurt, jams berries, grapes, apples, roses, red cabbage, sweet potato orange-red Anthocyanins baked goods, gravies, vinegars, syrups, colas, sauces heated sugarsbeige to brown Caramel candy, yogurt, ice cream, salad dressing, cake mixes red beets, cactus fruit redBetacyanins Naturally occurring colorants

56. Naturally occurring colorants cont. Used in Sources ColorsSubstance candy, dairy products, drinks, fruit fillings cochineal insects redCarmine meat products, cheese, butter, spice mixes, salad dressings saffron, tomatoes, paprika, corn, butter,red salmon, marigolds, marine algae, carrots yellow to orange to red Carotenoids (precurser of vit.A) dehydrated spinach green plant leaves green to olive green Chlorophylls

57. Naturally occurring colorants cont. Used in Sources ColorsSubstance flour, bread, pastries, cereals, dietary products vegetable leaves, milk, eggs, organ meats, malt yellowRiboflavin (vit.B 2 ) pickles, mustard, spices, margarines, ice cream, cheese, baked goods, soups, cooking oil, salad dressings Curcuma longa rhizome yellow curcumin