c


 

c c



 


 

   ! 

"# 

$$%&

The present paper deals with the chemistry, isolation, characterizationfor use in the
manufacture of foods as a natural food colorant. Marigold (TagetesErecta L, an ornamental
plant belonging to the composite family, has a rich source of natural antioxidant-Lutein. A
natural pigment, xanthophylls offer an alternative to synthetic dyes as a food colorant, due to
its non-toxicity. Lutein extracted from Tageteserecta and from other species of edible plants,
is already permitted for use as a food additive (E161b. The requested product has the same
specifications as the approved food additive. Results from human intervention studies with
foods high in lutein zeaxanthin, with lutein supplements using marigold as the lutein source
or with enteral tube feeding containing lutein, indicate that lutein from these sources is
bioavailable. The petitionerǯs proposed use of lutein is in foods for special medical purposes
(FSMPs at levels that would give rise to daily intakes of 0.5 to 2 mg lutein per day. This is
within the range of a regular dietary intake. Lutein extracted from the natural strains of
edible fruits and plants, grass, Lucerne (alfalfa and Tageteserecta (marigold flowers is
already permitted as a food additive (colour. Tageteserecta in FSMPs, is not of safety
concern under the proposed use levels which are in the range of the regular dietary intake of
lutein, provided that it is in compliance with the existing FDA and EU specifications of the
food additive.

'
%



Since the early civilizations and in the beginning of the food industry, natural pigments or
synthetic, were used to give an attractive presentation, perception of freshness, taste, and
quality of food. Saffron and other plant species were used to provide characteristic color and
flavor in food. Today, natural colorants are emerging globally due to the perception of its
safer and eco-friendly nature. Nowadays, a trend towards Dznaturalnessdz represents a
challenge for food manufacturers, because of its pharmacological applications. Synthetic
dyes received faster acceptability due to a wide range of applications in various fields like
food [1], cosmetic [2], and more importantly in textile [3 y 4] due to ease in dyeing, and
reproducibility in shades and overall cost factor. Natural foods colorants can be originally
present in the foodstuff, or may be added as an extract to enhance the natural color. Quality
of food is associated with many aspects -color, flavor, and texture, but color can be
considered the most important of them, because of its appealing nature. Color as well gives
the key to catalogue a food as safe, with good aesthetic and sensorial characteristics: the
undesirable colors in meat, fruits, and vegetables warn us about a potential danger or at
least of the presence of undesirable flavors, among other reactions.

The food industry is therefore, interested in gaining a better understanding of color

generation and color stabilization during the various steps of food processing. The processed
foods constitute 60- 65% of total food and the need for the food additives and its improved
shelf life is also increasing. There are some 2500 chemicals that function as food additives
that give rise to some 5000 trade name products on a worldwide basis [5]. However, the
natural pigments that are permitted for human foods are very limited, and the approval of
new sources is difficult, because the U.S. Food and Drug Administration (FDA considers the
pigments as additives, and consequently pigments are under strict regulations [6].

A high quality of food and beverages is vital to our physical and mental well being. Of all the
food additives in use, none gives rise to greater controversies than food colors. The readily
available coloring matter based on natural products is of considerable importance since the
United States have banned the use of synthetic coloring in foods. In ancient times tinted
amaranth has been used to extract the coloring matter, which is hydrophilic in nature and
was used for the dyeing of drinks, food and other products in Mexico, Bolivia and Ecuador
[7]. In India and Mexico, for facial rouge the woman used amaranth juice. This pigment
belongs to the group of betacyanines [8 y 9] and the betanine have been used as colorants in
many types of food [10]. Some of the important plant pigments are carotenoids,
Anthocyanins, and betalains. Carotenoids are compounds comprised of eight Isoprenoid
units (Ip whose order is inverted at the molecule center [6](Figure: 1.

Carotenoids are classified by their chemical structure as: (i carotenesthat are constituted by
carbon and hydrogen; (ii oxycarotenoids or xanthophylls that have carbon, hydrogen, and,
additionally, oxygen. Also, Carotenoids have been classified as primary or secondary.
Primary carotenoids group those compounds required by plants in photosynthesis ( p-
carotene, violaxanthin, and Neoxanthin, whereas secondary Carotenoids are localized in
fruits and flowers ( -carotene, p-cryptoxanthin, zeaxanthin, antheraxanthin, capsanthin,
capsorubin. Anthocyanins are the most important group of pigments, after chlorophyll,
which is visible to the human eye [11]. Chemically, Anthocyanins (Greek word anthosmeans
a flower, and kyanos, dark blue are flavonoids. Anthocyanins(Figure: 2 are substituted
glycosides of salts of phenyl-2- benzopyrilium (anthocyanidins. Chemically, betalain
definition embraces all compounds with structures based on the general formula (Figure 3
therefore; they are immonium derivatives of betalamic acid [12].

Figure. 1. Isoprene units &carotenoids structure.

Figure 2. Structure basic of anthocyanidin pigments in which Rx could be H, OH or OCH 3
depending of the considering pigment.

Figure 3. General formula betalain.

(A Betalamic acid moiety is present in all betalain molecules. (B Betacyanin or a
betaxanthin, depending on the identity of the R 1 and R 2 residues.

Carotenoids are abundant in fruits and plants and are widely used an antioxidant and may
be useful in the prevention of diseased including cancer. The consumption of lutein and
zeaxanthin reduces 40 % of the age related macular degeneration [13]. The xanthophylls
because of their yellow to orange-red coloration and natural occurrence in human foods,
also finds its use as a food colorant. Therefore there exist a high demand for the significantly
pure Xanthophyll that can be used as a food colorant and a nutrient supplement. Flowers
such as Tagetescomprise different species about 33 in number, helenium, helianthus,
sunflower, dandelion and many others. Of these, most concentratedsource of xanthophylls is
of the order 4500mg/lb [14] in the petals of TagetesErectaL (African marigold, Aztec
marigold, Zempasuchil. Marigold flower petals are a significant source of the Xanthophyll
and have a much higher concentration of this pigment compared to other plant materials
[15]. Marigold flower (TagetesErectaL is a hardy annual branching herb about 60 to 90 cm
tall and erect, grown in certain parts of India- Tamil Nadu, AndraPradesh , Karnataka as well
as in other parts of the World; and is extensively cultivated in temperate climate. It prefers
nourishing soil of pH 7.0 to 7.5 with good water -holding capacity and well-drained fertile
s s i sw s su i t xi s us i w s t iti
i i t R s i x si t su stitut s it w s
th wh i t u t th ts Eu u t th i w is x usi
us th i sti Dz dz i K I i th w t
Dz dz w t) Fi u ) u t its x ti i ti with th i t
w s

Fi u t iti ti Dz dz usi i w

i th i ti s u ti h ti u tu ti s th i w sh w
i ti s i u i w w i ht t t /h t )
th u h w is u t s x i s s t xi t t
th w s sist t s Ext ti stu i s t s t t w with
h x sh w th t w t s t i x th h s x t i s h h
whi h i tu ts s ti x th h s i s s th
t s us th is ti si

h i i t i w is ut i ) F Lut i h
xists i th w it tu u s i th t Fi u ) h Lut i st
t ti i sh i w s i s /K i ish w w st
/K i w w s w s t i ut ti s
Lut i st s th th i ht w s th h t t i s i th
t /K h t ti Lut i i si i t sh s i w s i
ish w t i ht w w t Lut i st s h
t t i th t /K w Lut i it t is th
st i th w h th st s ut i i ti i i th w i ist t
ist t it t it t st t ist t ) u ii xt t
i t s i t i i th h s i it t is t s
hth i t Lut i is st i t t xt i th
presence oflight, lutein undergoes isomerization resulting in color loss. Lutein structure
consists of conjugated bonds, which when react wi th the oxygen present in air, cause
oxidation to take place and lead to color loss. Oxidation products of xanthophylls are mono
and di-epoxides, carbonyls, alcohols etc. and extensive oxidation results in bleaching of
carotenoids pigment. To minimize color loss, it is safe to pack lutein-containing products in
tin or opaque containers.

Figure 5. Chemical structure of the xabthophylls lutein (p,
- carotene, top and zeaxanthin
(p,p- carotene, bottom.

Table 1. Composition of lutein fatty acid esters (%[16]

Enzymes like lipoxygenase hasten oxidative degradation, which occurs by direct

mechanisms. Enzymes first react with unsaturated or saturated fatty acids producing
peroxides, which react with lutein xanthophylls and lead to oxidative degradation.
DzBlanchingdz (98 oC/5 min exhibits an apparent increase in xanthophylls content due to
inactivation of lipoxygenase and also enhances pigment extraction [16].

Marigold carotenoids have potential as a natural food colorant. The status of marigold, as a
source of natural carotenoids, has been reviewed [14 y 15]. Temperature, pH, light, activity
in water is all factors, which affects the stability of the pigment [10]. Stability of Xanthophyll
pigment extracted from marigold has been studied by saponifying. Xanthophylls are usually
esterified which produces additional analyses complications and requires both separation
and identification. Saponification obtains less complex mixtures when only non-esterified
pigments appear. Another advantage of saponification is chlorophyll destruction in the
saponified samples [6]. Since commercial extracts are valued by their Xanthophyll and trans-
lutein content, concentration of lutein fatty acid esters in the extract can be enhanced by
purification.


( 

c 

('

Lutein is intended for use as a colouring agent and a nutrient supplement.

)(
  c 
Lutein is used as a colouring agent in foods such as baked goods and baking mixes,
beverages and beverage bases, breakfast cereals, chewing gum, dairy product analogs, egg
products, fats and oils, frozen dairy desserts and mixes, gravies and sauces, hard candy,
infant and toddler foods (other than infant formula, milk products, processed fruits and
fruit juices, soft candy, and soups and soup mixes. The intended food uses and use levels (2.0
Ȃ330 mg/kg are presented in (Tabla 2, [19].

()%  
Stability testing performed on the lutein products in commerce indicated that they are stable
at room temperature for a period of 12 months. Stability testing also was performed on
lutein in various food products, including pasta sauce, cereal bars and baked, ready-to-eat
cereal, which showed that they are stable at room temperature for a period of 12 months.
Lutein is not anticipated to react with other components of the food matrix or with
environmental constituents [19].


















































*+,   ,c c



(-#    
Lutein extracted from the natural strains of edible fruits and plants, grass, lucerne (alfalfa
and Tageteserecta, is authorised within the EU as food colouring agent (E161b (EC 1995.
Tagetes extract is authorised as a colour additive within the US (CDR 21 73.295. Tagetes
extract was evaluated as food additive by the JECFA [21] . The JECFA accepted its use but no
ADI was allocated. In 2004 JECFA established a group ADI of 0-2 mg/kg bw/day for lutein
and zeaxanthin as a food colour [22]. This lutein extract from Tageteserecta contains
relatively high levels (equal or higher than 70% of lutein and complies to the JECFA
specifications.

).c
c
/
c
c


)'.*
Several studies have investigated the bioavailability of lutein from marigold extracts by
assessing serum responses after a single oral dose of lutein. The effect of lutein
supplementation from marigold extracts on plasma responses has also been investigated in
several long-term intervention studies. A recent multi-centre, placebo-controlled
supplementation study investigated the effect of a lutein supplement containing 15 mg
lutein from marigold extracts during 20 weeks in 400 healthy male and female volunteers
from five different European countries aged 25-45 years [23]. Results showed a 5-fold
increase (by approximately 0.95 Ɋmol/L of plasma lutein concentrations, which reached a
plateau after 4 weeksof supplementation.In a study by Berendschot[24] the effect of daily
supplementation with 10 mg luteinderived from marigold during 12 weeks on macular
pigment density was investigated. This studyalso showed that plasma lutein concentrations
reached a plateau after 4 weeks. Mean plasma luteinconcentration increased from 0.18 to
0.90 Ɋmol/L within these 4 weeks and stayed at this levelduring the supplementation
period. This is a total increase of 0.72 Ɋmol/L.

In a study by Hiningeret al. (2001 175 healthy volunteers were supplemented with 15 mg
lutein orplacebo during 3 months. In the group receiving lutein supplementation, plasma
luteinconcentrations increased by 0.72 Ɋmol/L (from 0.22 ± 0.12 to 0.94 ± 0.13 Ɋmol/L and
LDL luteinconcentrations increased by 78 ng/mg LDL cholesterol.
Y
)'$* 
Mery little is known about the metabolism or degradation of lutein [25]. It is shown
thatlutein does not exhibit provitaminA activity [26].
Y
)(
# 
A 4-week pilot toxicity study was conducted in rats (Han Wistar to determine the oral
toxicityfollowing administration in the diet of a lutein product derived from marigold
flowers [27]. A compositional analysis of the product used identified 97 % of the
components andindicated that 84 % of the product consisted of the carotenoids lutein and
zeaxanthin at 79 and 5%concentration respectively. Waxes, palmitic acid/palmitate,
potassium and water made up theremainder [26]. Seven dose groups were used, including
the control (0, 2.6, 7.7,26.0, 77.3, 260 and 773.2 mg of lutein product/kg bw/ day. The study
was performed essentiallyaccording to OECD guideline 407 [28], with the exception that
hematology parametersmeasured did not include blood clotting time/potential. Necropsies
were performed after anovernight period without food after 4 weeks. Tissues were collected
for gross examination. Organweights were determined for adrenals, heart, kidney, liver,
ovaries, spleen, testes + epididymides,thyroids+parathyroids. According to OECD guidelines
the thymus and brain should have beenincluded as well (organ weight. Histopathology was
performed on liver, spleen, skin andmesenteric lymph nodes. According to the authors the
hematology and clinical chemistry analysis revealed sporadic, statistically significant effects
on several parameters compared with the control.
There was however no consistent dose response, changes were small, and there was no
correlationof the changes with any adverse treatment-related histopathology findings. These
changes weretherefore not considered to be treatment related or biologically significant by
the authors.

The authors concluded that oraladministration of a lutein product to rats at dose levels up to
773.2 mg/kg bw/day (highest doselevel used for 4 weeks did not result in test article
related toxicity and was well tolerated by therats.In addition a 13-week diet toxicity study in
rats (Han Wistar was conducted, consisting of threetreatment groups and one control group
(0, 2.6, 26 and 260 mg lutein (similar specifications asdescribed above/kg bw/day (Kruger
et al. 2002. This study was performed according to OECDguideline 408 [28]. It can
beconcluded that a dose level of 260 mg/kg/day (highest dose level tested for 13 weeks did
not resultin toxicity[29].

))$
The mutagenic potential of lutein product (source marigold, same material as used in the 4-
weekand 13-week study was investigated in the Ames test according to the guidelines laid
down inOECD 471 (OECD 1997 (Kruger et al. 2002. Two formulations were tested:
beadlets containing10% lutein product and lutein produ ct as such. For both formulations the
plate incorporationmethod and the pre-incubation method were used.
The concentration range used for beadlets containing 10% lutein was: 158-15,800 Ɋg/plate.
Theconcentration range used for lutein product was 15.8-1580 Ɋg/plate. The results
obtained were asfollows:Beadlet formulation: the maximal dose level of 15,800 Ɋg/plate was
not evaluated due toprecipitation. No increase in the number of mutant colonies was
observed with the beadletsformulation. The mutant frequencies of the controls were within
the range of historical controlvalues and data published in the literature. Lutein product: no
toxicity was apparent for any strain,except TA 102, which showed reduced growth, most
prominently in the absence of S9. No increas ein the number of mutant colonies was
observed with lutein product. The mutation frequencies ofthe controls were within the range
of the historical control values and the data published in theliterature.
Y
)-0 
Many studies have been conducted looking at the effects of dietary intervention with foods
high inlutein+zeaxanthin or lutein supplements. Some of these studies also used the
marigold source. Inthese studies effects on plasma levels were evaluated after lutein
ingestion which ranged from 0.4to 30 mg/day for a period up to 12 months [30-42]. With
the exception of the multi-centre Olmedillastudy [23] no side effects were reported in these
studies with respect to increasedcarotenoid consumption. However, these studies were not
designed to detect adverse effects. In theOlmedilla study 40% of the subjects in the Spanish
cohort only, supplemented for 20 weeks with 15mg lutein/day, using a lutein -rich marigold
extract showed carotenodermia, but no changes inbiochemical or hematological indices. The
petitioner indicates that no other examples are known inanimals or humans where lutein
supplementation alone, in a level higher than regular dietaryexposure, caused
carotenodermia.

)1
# 2 
High intake of carotenoid-containing foods or supplements is not associated with any toxic
side effects. As a result, the Institute of Medicine at the National Academy of Sciences did not
establish a Tolerable Upper Intake Level (UL for carotenoids when it reviewed these
compounds in 2000 [20].

-% c
%&

-'%
Lutein is approved for use in supplements under DSHEA. The FDA is being petitioned for
GRAS status of lutein for food supplementation. Lutein, responsible for the yellow color in
vegetables such as corn, is present in high amounts in foods like spinach and turnip greens.
Lutein cannot be produced in the body. It must be obtained through the diet [43, 44].

-'(% 
Officially notified the US Food and Drug Administration (FDA that natural lutein esters are
GRAS and can be used to fortify a variety of foods. As the first and only lutein ester supplier
to carry out such voluntary notification, is clearly demonstrating its commitment to
providing safe and efficacious ingredients to de food industry [44].

Food group Carotenoids Lutein esters Active Lutein

Esters Mg/serving equivalent
mg/serving mg/serving
Baked goods and baking mixes 4.3 4.0 2.2
Soy milk 3.2 3.0 1.7
Beverages and beverages powders 4.3 4.0 2.2
Frozen dairy desserts and mixes. 2.15 2.0 1.1
Processed fruit and vegetables products 4.3 4.0 2.2
Eggs products and eggs substitutes 4.3 4.0 2.2
Breakfast cereals (ready to eat and hot 4.3 4.0 2.2
Fats and oil 3.2 3.0 1.7
Hard candy 2.15 2.0 1.1
Fruit snacks 2.15 2.0 1.1
Dairy products 6.45 6.0 3.3

Dietary supplements: natural lutein esters are sold under the dietary supplement Health and
Education Act of 1994 (DSHEA which allows manufacturers to describe the supplementǯs
effects on Ǯstructure or functionǯ of the body or the Ǯwell-beingǯ achieved by consuming the
dietary ingredient[44].

-')%2  
According to EU directive 94/36/EC OF JUNE 1994  lutein and lutein esters are permitted
under E161b to be used in various food products as a colorant. The directive list the
following maximum levels in certain food products:
 3  $#c
Non-alcoholic flavored beverages 100ppm
Confectionery 300 ppm
Decoration and coatings 500 ppm
Fine baked goods 200 ppm
Ice cream 150 ppm
Flavored processed cheese 100 ppm
Desserts including flavored milk products 150 ppm
Sauces and seasonings 500 ppm
Fish paste 100 ppm
Salmon substitutes 500 ppm
Smoked fish 100 ppm
Extruded savory snack products 200 ppm
Meal replacer 50 ppm
Soup 50 ppm

Natural lutein esters meet the product specification outlined in the EU directive 95/45/EC
for specific purity criteria of food colorants (amended by 1999/75/EC/,2001/50/EC and
2004/47/EC [43].

-')'
As legislation on functional food has yet to be harmonized in the EU, Lutein esters as food-
fortifying ingredients are currently subject to country-specific laws and regulations [43].

-')( 22 
Lutein ester products have been used for human consumption in food supplements in the
European union prior to May 15, 1997. Therefore natural lutein esters are no considered
Novel in food supplements [43].

-'))* 2 

On April 25 th, 2006 a codex alimentarious additives committee decided to retain the current
specifications for lutein esters under E161b (i describing them as lutein from
Dztageteserectadz with functions DzColour and Nutritional Supplementdz [43].

-)2  
Letter from the European Commission to the Chairman of the Scientific Committee on Food
on commission request for a specific opinion on the evaluation of a number of substances
added for specific nutritional uses in foods for particular nutritional uses. SANCO
D4/AN/dlc- D(2003440384 Submission on behalf of IDACE, in reference to Commission
Directive 2001/15/EC on substances that may be added for specific nutritional purposes on
foods for particular nutritional uses. Author: Ree E, Title: Safety dossier for lutein propos ed
for use in Food for Special Medical Purposes. Wageningen, 2003.

1
 

Lutein, obtained by solvent extraction of species of edible fruits and plants, grass, lucerne
(alfalfaand Tageteserecta (marigold flowers, is authorized within the EU as food colouring
agent (E161b(EU 1995. The requested product complies with the specifications of the
approved food additive.Therefore, the use foreseen is in foods for special medical purposes
(FSMPs in this case for enthrallsips and tube feeds. According to the petitioner the
quantities of lutein to be added to FSMPproducts will be in the range 0.5 -2 mg/day of lutein.
For lutein this means use levels that give riseto daily intakes between 0.5 and 2 mg per day.
This is within the range of a regular diet, since thedaily intake of lutein as such is, based on
published literature, estimated to vary from 0.8 - 2.5 mgper day.Results from human
intervention studies with foods high in lutein and zeaxanthin or luteinsupplements using
marigold as the lutein source, indicate that lutein from these sources isbioavailable.In a 13-
week toxicity study in rats using a marigold extract, no adverse effect was observed at
thehighest dose tested (equivalent to 200 mg/kg bw/day.Lutein product, extracted from
marigold flowers, did not have mutagenic potential in the Ames test.Although the majority of
human studies have not been designed to assess the safety of lutein, theyhave not revealed
adverse effects. There are case reports of skin discolouration upon high intakes oflutein-rich
marigold extract.

+

c  %
$$
 

Results from human intervention studies with foods high in lutein plus zeaxanthin, with
luteinsupplements using marigold as the lutein source or with enthrall tube feeding
containing lutein,indicate that lutein from these sources is bioavailable.Lutein extracted
from the natural strains of edibable fruits and plants, grass, Lucerne (alfalfa
andTageteserecta (marigold flowers is already permitted as a food additive. Based on that,
the Panelconcluded that the use of lutein, obtained as an extract from Tageteserecta and
from the naturalstrains of edible fruits and plants, grass, lucerne (alfalfa and Tageteserecta
in FSMPs, is not ofsafety concern under the proposed use levels which are in the range of the
regular dietary intake oflutein, provided that it is in compliance with the existing EU
specifications of the food additive.The Panel is not able to evaluate the general use of lutein
in foods for particular nutritional usesince no information was provided on proposed uses
and use levels others than FSMPs.


4%%
 

[1] Torgils, F, Luis, C, Oyvind, M.A. (1998. Colour and stability of pure anthocyanins
influenced by pH in cluding the alkaline region. Food Chemistry, 63(4: 435Ȃ440.
[2] Calnan, C.D. (1976. Quinazoline yellow SS in cosmetics. Con tact Dermatitis, 2(3, 160-
166.

[3] Savarino, P, Miscardi, G, Quagliotto, P, Montoneri, B.E. (1999. Reactivity and effects of
cyclodextrins in textile dyeing. Dyes and Pigments, 42(2:143Ȃ147.

[4] Paisan, K, Aroonsiri, S, Nontalee, C. (2002. Thermodynamics of adsorption of laccaic acid

on silk, 53(2: 179 Ȃ185.

[5] Scotter, M.J, Castle, L. (2004. Food Additives and Contaminants, 21: 93 Ȃ124.

[6] Delgado-Margas.F, Paredes-Lopez, O, Jimenez, A.R. (2000. Natural Pigments:

Carotenoids, Anthocyanins, and Betalains Ȅ Characteristics, Biosynthesis, Processing, and
Stability. „ritical Reviews in Food Science and Nutrition, 40(3:173Ȃ289.

[7] Gladis Ester Scoles, Silvia HaydeePattacini, Guillermo Federico Covas. (2000. Separation
of the pigment of an Amaranth. Molecules, 5: 566,567.
[8] Attoe, E. L. & von Elbe, J. H. (1985. Oxygen involvement in betanine degradation: effect of
antioxidants, ]ournal of Food Science., 50: 106Ȃ110.
[9] Mabry, T.J; Drieiding.A.S. (1968. The Betalaines. In Recent Advances in Phytochemistry,
(1968.
[10] Pasch, J. H. and von Elbe, J. H.. (1979. Betanine stability in buffered solutions containing
organic acids, metal cations, antioxidants, or sequestrants, ]ournal of Food Science, 44: 72Ȃ
74,81.

[11] Harborne, J. B., Marbry, T. J., And Marbry, H., (1988, The Flavanoids .London. Chapman
& Hall.

[12] Strack, D., Steglich, W., Wray, M., (1993. Betalains. In: Methods in Plant Biochemistr y
Mol. 8, Academic Press, Orlando, 421Ȃ450.

[13]Seddon. (1994. ]ournal of American Medical Association, 272 (18: 1413-1420.

[14] Merghese, J. (1998b. Focus on xanthophylls from Tagetes Erecta L the giant natural
color complex-II. Indian Spices 34(1&2: 13Ȃ16.
[15] Merghese, J. (1998a. Focus on xanthophylls from TagetesErecta L the giant natural
complex-I. Indian Spices 33(4: 8Ȃ13.
[16] Sowbhagya. H. B, S. R. Sampathu, Krishnamurthy. (2004. Natural Colorant from
Marigold-Chemistry and Technology Food reviews International, 20(1: 33Ȃ50.
[17] Gregory, G.K.; Chen, T.S.; Philip, T. (1986. Quantitative analysis of lutein esters in
marigold flowers by high performance liquid chromatography. ]ournal of Food Science, 51:
1093Ȃ1094.
[18] Gau, W., Plosche, H. J ., Wunsche, C. (1983. Mass spectrometric identification of
xanthophyll fatty acid esters from Marigold flowers (TagetesErecta obtained by high
performance liquid chromatography and counter current distribution. ]ournal of
„hromatography, 262: 277Ȃ284.
[19] Lutein from TageteserectaChemical and Technical Assessment (CTAFirst draft
prepared by Richard Cantrill© FAO 2004Y
[20] Agarwal S, Rao AM. Carotenoids and chronic diseases. Drug Metabol Drug Interact
2000;17(1-4:189-210 2000. PMID:15130.
[21]JECFA (2001 JECFA food additive specification Tagetesextract. FNP 52 Add 9,
(http://apps3.fao.org/jecfa/additive_specs/docs/9/additive-0839.htm
[22]JECFA (2004 Evaluation of certain food additives.WHO Technical Report Series
928.WHO Geneva 2005.
[23] Olmedilla B, Granado F, Southon S, Wright AJA, Blanco I, Gil -Martinez E, Man den Berg H,
Thurnham D, Corridan B, Chopra M, Hininger I. (2002 A European multicentre,
placebocontrolled supplementation study with alpha -tocopherol, carotene-rich palm oil,
lutein or lutein: analysis of serum responses. ClinSci (Lond 102:447 -456.
[24] Berendschot TT, Goldbohm RA, Klopping WA, van de Kraats J, van Norel J, van Norren D
(2000 Influence of lutein supplementation on macular pigment, assessed with two objective
techniques. Invest Ophthalmol Mis Sci; 41:3322 -3326.
[25] Parker RS (1996 Absorption, metabolism, and transport of carotenoids.Faseb J 1996;
10:542-551.
[26]. Man Mliet T, van Schaik F, Schreurs WH, van den Berg H (1996 In vitro measurement of
betacarotene cleavage activity: methodological considerations and the effect of other
carotenoids on beta-carotene cleavage. Int J MitamNutr Res 66:77-85.
[27]Landrum JT, Bone RA, Joa H, Kilburn MD, Moore LL, Sprague KE (1997 A one year study
of the macular pigment: the effect of 140 days of a lutein supplement. Exp Eye Res 65:57 -62.
[28]OECD (1997OECD guideline 471: Bacterial reverse mutation test.
[29]Kruger C, Murphy M, DeFreitas Z, Pfannkuch F, Heimbach J (2002 An innovative
approach to the determination of safety for a dietary ingredient derived from a new source:
case study using a lutein product product. Food ChemToxicol 40:1535 -1549..
[30]Broekmans WM, Klöpping-Ketelaars IA, Schuurman CR, Merhagen H, Man den Berg H,
Kok FJ, Man Poppel G (2000 Fruits and vegetables increase plasma carotenoids and vitamins
and decrease homocysteine in humans. J Nutr 130:1578-1583.
[31]Castenmiller JJM, West CE, Linssen JPH, Man het Hof KH, Moragen AGJ (1999 The food
matrix of spinach is a limiting factor in determining the bioavailability of beta-carotene and
to a lesser extent of lutein in humans. Journal of Nutrition 129:349 -355.
[32]Handelman GJ, Nightingale ZD, Lichtenstein AH, Schaefer EJ, Blumberg JB (1999 Lutein
and zeaxanthin concentrations in plasma after dietary supplementation with egg yolk. Am J
ClinNutr 70:247-251.
[33]Hininger IA, Meyer-Wenger A, Moser U, Wright A, Southon S, Thurnham D, Chopra M,
Man Den Berg H, Olmedilla B, Favier AE, Roussel AM (2001 No significant effects of lutein,
lycopene or beta-carotene supplementation on biological markers of oxidative stress and
LDL oxidizability in healthy adult subjects. J Am CollNutr 20:232 -238.
[34]McEligot AJ, Rock CL, Shanks TG, Flatt SW, Newman M, Faerber S, Pierce JP (1999
Comparison of serum carotenoid responses between women consuming vegetable juice and
women consuming raw or cooked vegetables. Cancer Epidemiol Biomarkers Prev 8:227-231.
[35]Micozzi MS (1992 Plasma carotenoid response to chronic intake of selected foods and
betacarotene supplements in men. American Journal of Clinical Nutrition 55:1120-1125.
[36]Muller H, Bub A, Watzl B, Rechkemmer G (1999 Plasma concentrations of carotenoids
in healthy volunteers after intervention with carotenoid-rich foods. Eur J Nutr 38:35-44.
OECD (1998 OECD guideline 408: Repeated dose 90 -day oral toxicity study in rodents.
[37]Olmedilla B, Granado F, Blanco I, Rojas-Hildago E (1996 Evaluation of retinol, alpha -
tocopherol, and carotenoids in serum of men with cancer of the larynx before and after
commercial enteral formula feeding. Journal of Parenteral and Enteral Nutrition 20:145-149.
[38]Olmedilla B, Granado F, Gil-Martinez E, Blanco I, Rojas-Hidalgo E (1997 Reference
values for retinol, tocopherol, and main carotenoids in serum of control and insulin -
dependent diabetic Spanish subjects. ClinChem 43:1066 -1071.
[39]Rock CL. Platt SW, Wright FA, Faerber S, Newman M, Kealey S, Pierce JP (1997.
Responsiveness of carotenoids to a high vegetable diet intervention designed to prevent
breast cancer recurrence. Cancer Epidemiology, Biomarkers and Prevention 6:617-623.
[40]Rock CL, Thornquist MD, Neuhouser ML, Kristal AR, Neumark-Sztainer D, Cooper DA,
Patterson RE, Cheskin LJ (2002 Diet and lifestyle correlates of lutein in the blood and diet.
Journal of Nutrition 132:525S -530S.
[41]Roodenburg AJ, Leenen R, van het Hof KH, Weststrate JA, Tijburg LB (2000 Amount of
fat in the diet affects bioavailability of lutein esters but not of alpha-carotene, beta-carotene,
and vitamin E in humans. Am J ClinNutr 71:1187 -93.
[42]Man het Hof KH, Brouwer IA, West CE, Haddeman E, Steegers-Theunissen RPM, Man
Dusseldorp M, Westrate JA, Eskes TKAB, Hautvast JGAJ (1999 Bioavailability of lutein from
vegetables is 5 times higher than that of beta-carotene. Am J ClinNutr 70:261-268.
[43]El portal de la Union Europeahttp://europa.eu/pol/food/index_es.htm
[44]Food and Drugs Administration http://www.fda.gov/