Introduction

Natural Occurrence Of Carotenoids

Carotenoids are lipid secondary metabolites that play essential roles in plants and are also relevant compounds from a nutritional standpoint. (Etoh et al., 2000; Grobush et al., 2000). They attract much attention due to their proposed antioxidant properties but also as (mainly orange-red) natural pigments abundant in many fruits and vegetables (like oranges, tomatoes and carrots flowers etc.) that constitute an important part of the human diet. (Edge et al., 1997; Meléndez-Martínez et al., 2014).

Decades of research on carotenoids has improved our understanding of the role of these ubiquitous pigments, which have emerged as important players exerting a protective role against diseases associated with aging, including cancer, cardiovascular disease, cataracts, and age-related macular degeneration (Bowen et al., 2015; Bermudez et al., 2005; Pantavos et al., 2015). They have been studied for a number of years because of their diverse roles in photobiology, photochemistry and photo medicine (Pryor et al. 2000). Carotenoids are also added as colorants to many manufactured foods, drinks, and animal feeds, either in the forms of natural extracts (e.g annatto, paprika or marigold extracts) or as pure compounds manufactured by chemical synthesis (Kiokias et al., 2009^a).

Carotenoids are often described as provitamins A, as this particular vitamin is a product of carotenoid metabolism. The distribution of carotenoids among the different plant groups shows no obvious pattern (Coultate, 1996). b-Carotene is the most abundant in leafy vegetables, though the colour is masked by its co-existence with chlorophyll, and this carotenoid has the highest vitamin A activity. Zeaxanthin, a-carotene and antheraxanthin are also present in small amounts. In the tomato, lycopene is the major carotenoid, while fruits contain varying proportions of cryptoxanthin, lutein and antheraxanthin. (Van den Berg et al., 2000). Furthermore, some animals owe their colour to carotenoids, especially birds (yellow and red feathers) and fish (salmon), whereas complex formation with protein may modify their colour to blue or green (Ribayamercado et al., 2000).  The total carotenoid content of different foods materials is given in table-2 (Kiokias, 2002).

Although close to 600 carotenoids have been identified in nature only 50 possess provitamin A activity and about 40 are present in a typical human diet, whereas of these only 14 and some of their metabolites  have been detected  in blood and tissues (Akoh & Min., 1997).

Carrot, tomato and papaya represent important dietary sources of β-carotene and lycopene (Schweiggert et al., 2015). A normal and varied food supply provides 1000-4000 mg of carotenoids daily (Mangels et al., 1993). Actually, the daily intake of carotenoids is generally more variable than the intake of protein, fat, and carbohydrates as characterized by large fluctuations. For example, 100g servings of cooked mustard greens, spinach and broccoli, provide very different amounts of carotenoids as shown in Table-1 (Kiokias, 2002).

Carotenoids are believed to play a number of vital roles in the physiology of the plant kingdom, and they are ultimately involved with photosynthesis, the fundamental life-sustaining reaction on the planet (Kritchevsky 1999; Kiokias et al., 2008^a). Moreover, there is a large body of evidence that certain natural carotenoids (e.g. lycopene, lutein and zeaxanthin etc.) possess various antioxidant functions exhibiting both a technological action against lipid oxidation and a protective role in delaying the onset of chronic disease (Krinsky 2001; Mares-Perlman et al., 2002).

Structure And Biosynthesis

Most carotenoids are 40-carbon terpenoids having isoprene as their basic structural unit (Khachik et al., 1997). A general subdivision is into:

(i) “carotenes” which are strictly hydrocarbons (a– and b-carotene, lycopene) and    (ii) “xanthophylls” (lutein, bixin, capsanthin etc), which contain polar ends groups reflecting an oxidative step in their formation (Bohm et al., 1999;  ).

The simplest  carotene is lycopene, from which a– and b-carotene, derive by cyclisation at the end of the chain (Faure et al., 1999).  The xanthophylls arise initially by hydroxylation of the carotenes, and their subsequent oxidation reactions lead to the formation of epoxides such as antheraxanthin (Kovary et al., 2001). The heat treatment of carotenoids sometimes leads to their isomerisation. For instance, a– and b-carotene that differ only in the position of a double bond in the cyclic end-group can  both show further cis/trans-isomerism along the terpene chain. Most naturally occurring carotenoids possess a trans configuration for all conjugated double bonds. (Madhovi et al., 1996).

A recent body of scientific evidence focused on biosynthesis of natural carotenoids (Liu et al., 2013). The carotenoid biosynthetic pathway serves manifold roles in plants (related to photosynthesis, photoprotection etc.) and also produces compounds that impact human nutrition and metabolic products that contribute to fragrance and flavor of food and non-food crops (Shumskaya and Wurtzel, 2013).

According to Britton (1995), the conjugated polyene chromophore determines the light absorption properties, and hence colours, having also a strong influence on the physicochemical properties of the molecule. Structural features such as size, shape, and polarity are essential determinants of the ability of a carotenoid to fit correctly into its environment to allow it to function (Kiokias & Gordon 2004; Mortensen & Skibsted.,1997). Carotenoids in plants are generally water insoluble and are associated with lipids in chloroplast cells. Because of their water-insolubility they do not leach out when the vegetables are prepared and cooked, nor do they change colour markedly with heat or pH, particularly if the chloroplast cells remain relatively intact (Rodriguez-Concepcion and Stange, 2013). On the other hand, they are slightly soluble in oils at room temperature and in non-polar organic solvents, such as chloroform and acetone (Simpson, 1983). In addition, it has been reported that regardless of the food conservation method used, carotenoids undergo a slow degradation on storage, with a loss depending on matrix and storage conditions (Meissonnier, 1983; Bohm et al., 1997).

Carotenoids are synthesized in nature by plants and many microorganisms (Bai et al., 2015). Animals can metabolize them in a characteristic manner, but they are not able to synthesize them. Being terpenoids, carotenoids are synthesized form the basic C5- terpenoid precursor, isopentyl diphosphate (XVII, Figure-2, Kiokias, 2002).  This compound is converted to geranyl-geranyl diphosphate(XVIII). Its dimerisation leads to phytoene and the stepwise dehydrogenation via phytofluene (X), zeta-carotene (XXI) and neyrosporene (XXII), and gives lycopene (I). Subsequent cyclisations, dehydrogenations, and oxidation reactions, lead to the other naturally occurring carotenoids.Technological advances have made possible the synthesis, at reasonable prices, of carotenoids with well-controlled, reproducible colours, without quality variations, and in a volume that can be scheduled to meet the needs of the food industry (Basu et al., 1999).  Carotenoids have been extensively used by the food industry as colorants in the production of many foods. Micropulverised dispersions of carotenoids are used in coloring fat based foods such as margarine, butter, shortenings, cheese, and french dressings. Water-dispersible forms of carotenoids have been developed for the coloring of water-based foods such as orange-type beverages, cake mixes, puddings, dried and canned soups (Boileau et al., 1999).

Absorption And Transport Of Dietary Carotenoids

The absorption and transport processes of carotenoids are quite complex and to a large degree not well understood. Absorption is defined as a movement of dietary carotenoids, or their metabolites to the lymphatic or portal circulation (Erdman et al., 1993). Several processes are necessary for optimal absorption to occur: i) sufficient digestion of the food matrix to release carotenoids, ii) formation of lipid micelles in the small intestine, iii) uptake of carotenoids by intestinal mucosal cells, and iv) transport of carotenoids or their metabolic products to the lymphatic or portal circulation (Berni et al., 2015). After absorption through passive diffusion, carotenoids follow the chylomicrons metabolism, they are taken up by the liver and released in the blood stream in lipoproteins (Faure et al., 1999). In the fasted state, the hydrocarbon carotenes are carried by VLDL and LDL lipoproteins, residing in the hydrophobic core of the particles, while the more polar xanthophylls are found mainly in HDL, closer to the surface (Krinsky et al., 1988). As depicted in figure 3 (Kiokias et al., 2002). carotenoids accumulate or are stored in tissues. It is assumed that at least in the liver, beta-carotene and other provitamin A carotenoids would be available for conversion to Vitamin A (Kopec et al., 2015). The delivery of carotenoids to extra-hepatic tissue is accomplished through the interaction of lipoprotein particles with receptors and their further degradation by extra-hepatic enzymes such as lipoprotein lipase. Adipose tissues and liver appear quantitatively to be the main storage sites, whereas adrenal gland, kidney and testes also contain a high per gram concentration. It is found, that mild cooking and additional ingestion of dietary fats improves carotenoid absorption. This is likely to be due to the release of carotenoids from cellular components of the plants upon heating and the formation of carotenoid-containing micelles from dietary fat, which facilitate carotenoid absorption in the gut (Khachik et al., 1997).

Dietary lipids have been shown to increase bioavailability of provitamin A carotenoids from a single meal, but the effects of dietary lipids on conversion to vitamin A during absorption are essentially unknown. Kopec et al. (2014) highlighted the importance of provitamin A carotenoid consumption with a lipid-rich food such as avocado for maximum absorption and conversion to vitamin A, especially in populations in which vitamin A deficiency is prevalent. Berni et al., (2015) has recently examined the bioaccessibility of Provitamin A carotenoids in home cooked and commercially processed orange fleshed sweet potato.

It is generally accepted that the serum carotenoid concentration reflects the immediate dietary intake. Although, this is a major factor, others may be involved, such as sex, smoking and drinking habits, seasonal variations and geographic origin (Olmedilla et al., 1994).

The concentration of major carotenoids, found in human serum and tissues are given in table 1.3 (Basu et al., 1999). It has been found that 2 μg of β-carotene is equivalent to 1 mg of retinal in its ability to cure vitamin A deficiency in humans. The RDA (Recommended Dietary Allowances) however, uses conversion factors of 6 to1 and 12 to 1 for β-carotene and other provitamin A active carotenoids respectively, recognizing that compared to more purified forms, dietary carotenes are much more poorly absorbed (Simpson et al., 1985). Therefore for products containing β-carotene and other provitamin A carotenoids:

Total RE (retinal equivalent): (μg β-car/6) + (mg other provitamins A/12)

The RDA for men is 900 μg/day, and for women 700 μg/day, except in pregnancy (770 μg/day) and lactation (1.300 μg/day).

The US Institute of Medicine (2000) has introduced a new term, “retinal activity equivalent” or RAE, to express the vitamin A activity of carotenoids.

1 RAE = 1 μg dietary or supplemented vitamin A =2  mg β-carotene in oil= 12 μg dietary β-carotene =24 μg other provitamin A carotenoids in diet

The estimated RAE is 625 μg/day for men and 500 μg/day for women.

Consumption of foods rich in beta-carotene is recommended by scientific and government organizations such as the U.S. National Cancer Institute (NCI) and the U.S. Department of Agriculture (USDA); these dietary guidelines recommend a dietary intake of 3 mg to 6 mg beta-carotene/day, which is associated with a lower risk of chronic diseases.

It should be noted, however, that the European Food Safety Authority (2006) and the U.S. Food and Nutrition Board (2000) have decided that the existing evidence is insufficient to establish a recommended dietary allowance (RDA) or adequate intake (AI) for beta-carotene and other carotenoids. In most European countries, the recommended intake is based on the assumption that 4.8 mg beta-carotene is needed to meet the requirement of 800 micrograms vitamin A (conversion factor 6). From epidemiological studies it can be concluded that a plasma level of 0.4 micromole/liter beta-carotene should be aimed at in order to benefit from the preventive health potential. This can be achieved with 2–4 mg/day (Biesalski et al., 1997).

Natural Plant Extracts And Carotenoid Analysis

A certain body of literature has focused on the analysis and functional properties certain natural extracts of carotenoids such as paprika, lycopene, lutein-rich extracts (Kiokias & Gordon, 2003^b; Kiokias et al., 2008^b).

A few of the most common -idustrially manufactured-extracts of natural Carotenoids are listed below:

1. Natural extract of carotenes: Carrot extracts, carrot oil, and palm oil related extracts are available in the market and their main components are a- and b-carotenes (Kiokias and Gordon, 2003^a). Purified crystalline products, dispersions of microcrystals in oil and carrot oil are also commercially produced.
2. Natural tomato Lycopene extract: Lycopene is a tetraterpenic C40 carotenoid that absorbs light in the red region being thereby responsible for the color of tomato and watermelon (Gann et al, 2012). For industrial uses natural lycopene is readily purified from tomato processing wastes (Wandai & Shaikly, 1985, Silva et al., 2013).
3. Refined Marigold extracts: Refined marigold extract is a natural yellow food colorant produced from marigold flowers  (Targeta Erecta) and a stable source of the carotenoid lutein, which is a normal constituent of  human plasma and retina (Vargas & Lopez, 1996).
4. Paprika oleoresin extract: Extracts of paprika (Capsicum annuum), rich in capsanthin carotenoids, are the oldest and most important natural carotenoid food colours, used as dry powder or oleoresin of paprika which is the oil extract of coloring and flavoring components from the pods (Pérez-Gálvez and Mínguez-Mosquera, 2004; Giuffrida et al., 2013) Oleoresin of paprika, unlike ground paprika, is a liquid product completely soluble in oils and therefore does not impart specks or vegetable tissues to the food product. (Mordi, 1993). A great advantage of the oleoresin is the possibility of standardizing the color of the oil extract since the paprika depends on various factors such as drying temperature, moisture content and storage conditions (Deli et al., 1992). Topuz et al. (2011) have recently examined the influence of different drying methods on carotenoids and capsaicinoids of paprika.
5. Natural preparations of Annatto seeds: The term annatto includes a series of coloring preparations consisting of carotenoid-type pigments; all based on extracts of the seed of the tree Bixa orellana which grows abundantly in the tropics (Chisté et al., 2011). Bixin is the main component of oil-soluble preparations, and norbixin of the water-soluble products. Norbixin powder is obtained from fresh annatto seeds by means of alkaline extraction following by a precipitation of the colorants with a mineral acid. The extract is then filtered pressed and dried at controlled temperature in order to permit its preservation (Scotter et al., 1998).  It produces orange solutions, suitable to color foods including cheese products, butter, margarine and salad dressing. The major coloring component of annatto is the apo-carotenoid 9-cis-bixin, the solubility of which is commercially achieved by heating a preparation of the seeds in oil to a maximum temperature of 130˚C in vacuo. Under these conditions, 9 cis bixin undergoes isomerisation to produce oil-solutions containing variable proportions of the pigment dependent on extraction temperature and time (Scotter,1995).

The main carotenoid pigments that are present in a range of common natural extracts are given in Table-4  (Kiokias et al., 2002).

Classical method of analysis of carotenoid pigments using column chromatography, paper chromatography and TLC potentiate isomerisations and transformations during the separation (Philip & Chen, 1988). Carotenoid extracts from natural sources are usually saponified to remove chlorophylls and unwanted lipids and to hydrolyse carotene esters (Khachik & Beecher, 1988). Organic solvents extract carotenoids, whereas fatty acids that precipitate as soaps and glycerol are retained in the aqueous phase. In addition, High Performance Liquid Chromatography (HPLC) is the analytical method of choice for separation, quantification and structural characterization of the naturally occurring and synthetic carotenoids as well as for their most important metabolic products (Rodriguez-Amaya, 2015). A body of research (Kiokias & Gordon 2003^a; Kiokias & Oreopoulou 2006) has focused on the analysis of natural extracts by Reversed Phase HPLC for the identification of the containing pigments. Additionally, the introduction of the photodiode array detector has facilitated the identification of carotenoids after HPLC separation by using spectral characteristics (Shoefs et al., 1995).

In food analysis spectrophotometry has been widely applied to the determination and quantification of carotenoids following the removal of interfering substances by liquid chromatography. The total carotenoid content of specific natural extracts (paprika, annatto, marigold etc.) has been determined by visible spectroscopy measuring total absorption at a specific wavelength and using absorptivity values reported in the literature (Kiokias et al., 2009^a). Solutions of the various carotenoid extracts (1mg/100 ml) were prepared after saponification and a spectrum of each one (200-500 nm) in a selected solvent was obtained. The specific absorbance, at the l_max of the major carotenoid for each extract was calculated, and compared with absorptivity values reported in the literature. (Hart & Scott, 1995). The l_max values of the major natural carotenoids (in n-hexane), with their absorptivityare also provided in table-4. A large body of scientific evidence suggests that carotenoids scavenge and deactivate free radicals both in vitro and in vivo, whereas a few researchers claimed that they can also act as prooxidants by accelerating rather than retarding the oxidative process (Khachik et al., 1997; Bub et al., 2000, Matos et al., 2000; Kiokias & Varzakas, 2014).  Carotenoids can also act as chemical quenchers undergoing irreversible oxygenation. The molecular mechanisms underlying these reactions are still not fully understood, especially in the context of the anti- and pro-oxidant activity of carotenoids, which, although not synthesized by humans and animals, are also present in their blood and tissues, contributing to a number of biochemical processes (Tessa et al., 1995; Fiedor, and Burda, 2014).  The antioxidant activity of carotenoids is a direct consequence of the chemistry of their long polyene chain (Farombi & Burton 1999; Boileau et al., 1999): a highly reactive, electron–rich system of conjugated double bonds susceptible to attack by electrophilic reagents, and forming stabilized radicals (Mortensen & Skibsted, 1997; Bast et al., 1998; Kiokias et al., 2008^b). Therefore, this structural feature is mainly responsible for the chemical reactivity of carotenoids towards oxidizing agents and free radicals, but also other factors such as oxygen pressure (Jorgensen and Skibsted, 1999) and synergistic effect with other natural compounds may determine their antioxidant action or even prooxidant character in certain model systems (Mordi et al; 1993; Kiokias & Gordon 2004, Krinsky 2001; Palozza, 1998). In the last decade, there is an increasing interest of a few researchers in  investigating  the effect of natural carotenoid extracts both in vivo following dietary supplementation (Kiokias and Gordon 2003; Stahl & Sies, 2005; Hofer et al., 2014) and in vitro in oil based systems (Dimakou and Oreopoulou, 2012; Viuda-Martos et al., 2012; Kiokias et al., 2009^b). Further experimental evidence of the antioxidant potential of certain carotenoid extracts following their incorporation in olive oil and olive oil based oil-in-water emulsions along with aspects of analysis and identification of their carotenoid pigments, are discussed by Kiokias and Varzakas (2014).

Table 1: Carotenoid content of selected foods and vegetables (mg/100g) (Kiokias et al., 2002)

Table 2: Total carotenoid content of selected food items (Kiokias, 2002)

Table 3: Concentration of Selected Carotenoids in Human Serum and Tissues (Basu et al., 1999)

Table 4: Common  natural carotenoid extracts (Kiokias, 2002)

Figure 1: Structure of the most common natural carotenoids (Kiokias & Oreopoulou, 2006).  Click here to View figure

Figure 2: Biosynthesis of natural carotenoids (Kiokias, 2002)  Click here to View figure

Figure 3:  Post-absorption transport of b-carotene to liver and extrahepatic tissues (Kiokias, 2002)  Click here to View figure

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