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Natural Colorant from Marigold-Chemistry and

Technology
a a a
H. B. Sowbhagya , S. R. Sampathu & N. Krishnamurthy
a
Central Food Technological Research Institute , Mysore, India
Published online: 16 Aug 2006.

To cite this article: H. B. Sowbhagya , S. R. Sampathu & N. Krishnamurthy (2004) Natural Colorant from Marigold-
Chemistry and Technology, Food Reviews International, 20:1, 33-50, DOI: 10.1081/FRI-120028829

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 FOOD REVIEWS INTERNATIONAL
Vol. 20, No. 1, pp. 33–50, 2004

Natural Colorant from Marigold-Chemistry

and Technology
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H. B. Sowbhagya,* S. R. Sampathu, and N. Krishnamurthy

Central Food Technological Research Institute, Mysore, India

ABSTRACT

Natural pigments offer an alternative to synthetic dyes, but for successful

application, an understanding of the chemical and physical properties of the
pigment is essential. With the growing legislative restrictions on the use of
synthetic colors, a reappraisal of natural plant pigments is taking place with a
view to use them as possible colorants in foods. For natural pigments to be
accepted as food colorants, legal sanction is a must. With the application of new
innovations, natural pigments can become more cost effective and increase their
competitiveness against certified dye and dye products. Marigold flowers, which
are yellow to orange red in color, are a rich source of lutein, a carotenoid pigment.
This pigment has acquired greater significance because of its antioxidant property
and for its eye health protection. Although marigold flower extract has been used
in veterinary feeds, the potential use of marigold as a natural food colorant has
not been exploited to the full extent due to the lack of information on its safety,
stability, and compatibility in foods. This article deals with the chemistry,
processing, and stability of the pigment and its applications.

Key Words: Marigold; Processing; Storage; Pigments; Natural colorant.

*Correspondence: H. B. Sowbhagya, Central Food Technological Research Institute,

Mysore-13, India; E-mail: sowbha@yahoo.com.

33

DOI: 10.1081/FRI-120028829 8755-9129 (Print); 1525-6103 (Online)

Copyright & 2004 by Marcel Dekker, Inc. www.dekker.com
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34 Sowbhagya, Sampathu, and Krishnamurthy

INTRODUCTION

Raw Material

Marigold (Tagetes erecta L.) is an ornamental plant belonging to the

Compositae family. It is grown in certain parts of India as well as in other parts of
the World. Marigold is widely grown as a border plant in gardens. Different varieties
and flowers are available in various shades of yellow, red, orange, dark orange, and
orange brown. The two common species of marigold, both annuals, are disting-
uished as African Aztec (T. erecta) and French marigold (T. patula) and are native to
Mexico and Gautemala. The African Aztec has large yellow or orange flower heads.
The French marigold has smaller single or double heads, usually with two tones of
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yellow or orange and red. The plant is native to Mexico and reported to be used in
traditional Mexican medicine. It is a hardy annual branching herb about 60 to 90 cm
tall and erect, and is extensively cultivated in temperate climate. It prefers a
nourishing soil of pH 7.0 to 7.5 with good water-holding capacity and well-drained
fertile sandy loamy soil as well as a sunny climate. Propagation is by seed, cutting, or
fresh stem. Leaves are strongly scented and pinnately divided, and leaflets are
lanceolate and serrated. Flowers are single to fully double with large-size globular
heads, and the color varies from lemon yellow to yellow, golden yellow, or orange.
Many Tagetes species yield aromatic essential oils, which are known as Tagetes
oils. The leaves are rich in oil. The yield of marigold flowers is about 30,000
kg/hectare (Wealth of India, 1976). Depending on the varieties, cultivar and
horticulture practices, the flower yield shows remarkable variations in number and in
flower weight, from 11 to 30 ton/hectare. Approximately 40% to 50% of the flower
consists of petals.
The main coloring component of the flower is lutein (C40H56O2), a carotenoid
pigment. Lutein naturally occurs in the acylated form. The lutein ester concentration
in fresh marigold flowers varies from 4 mg/g in greenish yellow flowers to 800 mg/g in
orange brown flowers (Gregory et al., 1986). Dark-colored flowers contain about
200 times more lutein esters than the light-colored flowers. Xanthophyll content
varies in the range of 4 to 5 g/lb. The concentration of lutein varies in different
shades of marigold flowers, viz., greenish yellow to bright yellow and orange brown
(Table 1). Total lutein esters have been reported to be in the range of 3.8 to 791 mg/g
of flower. Lutein palmitate is the major ester in the flower. The other esters of lutein
identified in the flower are dimyristate, myristate palmitate, palmitate sterate, and
distearate (Table 2). A purified extract of marigold petals mainly containing
xanthophylls dipalmitate is marketed as an ophthalmologic agent under the name
‘‘Adaptinol’’ (Gau et al., 1983).

CHEMISTRY OF PIGMENTS

Lutein, the major pigment responsible for the color in marigold, accounts for
60% of the total carotenoids present (Phillip and Berry, 1975). The carotenoid esters
in the marigold species are lutein, 70% to 90%; zeaxanthin, 10% to 25%; and a
small proportion of b-cryptoxanthin. Although not less than 17 components have
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Colorant from Marigold-Chemistry 35

Table 1. Concentration of lutein esters in marigold flowers.

Hunter color Lutein esters concentration (mg/g)

Observed Myristate Palmitate
color L A B Dimyristate palmitate Dipalmitate stearate Total

Green-yellow 46.7 1.9 25.9 — 1.53 1.82 0.48 3.83

Bright-yellow 41.0 11.1 22.2 — 2.74 5.29 — 8.03
Yellow 43.9 18.5 24.7 — 4.61 82.17 91.93 178.71
Orange 39.0 25.2 20.0 0.41 116.46 202.87 — 323.40
Orange 34.8 22.3 16.4 — 101.78 205.59 91.45 398.82
Dark-orange 38.8 29.1 19.6 48.79 245.08 373.98 1.40 869.25
Orange-brown 26.5 18.8 8.1 30.29 356.39 404.41 — 791.09
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Source: Adapted from Gau et al. (1983).

Table 2. Composition of lutein fatty acid esters (%).

Xanthophyll esters Ref. 3 Ref. 13

Dipalmitate 35.5 37.57

Dimyristate 12.6 11.57
Myristate palmitate 24.7 24.23
Palmitate-stearate 14.4 15.55
Distearate 2.4 3.63

Source: Adapted from Gau et al. (1983) and Helrich (1990).

been identified in Tagetes pigment complex, the contribution of constituents other

than lutein, zeaxanthin, and cryptoxanthin (Table 3) is insignificant. Lutein is a diol
of a-carotene and zeaxanthin is diol of b-carotene. Lutein is present in different
isomeric forms and in nature they occur as a mixture of trans (60%–90%) and cis
forms (10–40%). The different isomers of lutein present in marigold are 9-cis lutein,
13-cis lutein, 15-cis lutein, and all-trans lutein (Fig. 1). It is reported that trans
carotenoids are more effective and stable.
Lutein in natural form exists both in free and esterified forms. During heat
processing, a small fraction gets converted to cis form. Lutein changes from trans
form into its isomeric forms by the action of light, heat, oxygen, and acid. Both trans
and cis forms of lutein have biological value in maintaining health of the eye
(Anthony and Shankaranarayana, 2001). On hydrolysis, the pigments gain polar
property, which can be reversed by reesterifying with acetic anhydride/pyridine. The
status of unsaponified xanthophylls testifies that the hydroxyl groups are locked in
by acylation with fatty acids.
Techniques for extracting pigments from marigold flowers and the properties
of the pigment have been studied. Pigment was extracted with ethanol solution of
pH greater than 7 and recovered as a brown solid after distillation, condensation,
and precipitation. The solid was soluble in ethanol, methanol, cyclohexane and
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36 Sowbhagya, Sampathu, and Krishnamurthy

Table 3. Carotenoids of marigold.

Carotenoid Distribution (%)

Phytoene 2.4
Phytofluene 2.6
a-Carotene 0.1
b-Carotene 0.5
a-Zeacarotene 0.5
a-Cryptoxanthin 0.8
b-Cryptoxanthin 0.5
Lutein 72.3
Antheraxanthin 0.1
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Zeaxanthin 16.4
Neoxanthin 0.8

sparingly in water. Pigment was reported to have a bright yellow color at pH 7 with
color becoming lighter at lower pH and darker at higher pH (Zhang and Wang,
1994). The pigment has high heat (80–100
C for 3 min) and daylight stability. A
mixture of lutein dipalmitate and lutein dimyristate are soluble in hot corn oil (80
C)
to an extent of 35% w/w. Solubility of lutein esters in cold oil is less than 25%
(Phillip and Berry, 1975).

Stability of Pigments

Enhanced stability of xanthophylls are reported when the esters are partially
saponified followed by neutralization with a weak acid so the final product of about
pH 8 contains 10% to 20% by weight of unsaponified original xanthophyll esters.
Weak acids like acetic, propionic, or lauric have been used to neutralize the residual
alkali. Lutein esters upon saponification result in lutein, free of esters in trans form.
Better stabilization of marigold concentrate is achieved by adding an edible
vegetable fat (soybean oil, cotton seed oil) and then heating the mixture to at least
70
C. By employing drum-dying or spray-drying methods, colorant can be obtained
in the powder form. Addition of fat confers the beneficial effect of bringing
xanthophyll in better contact with the antioxidant because of their mutual solubility
in fat. Xanthophyll esters in marigold flowers are reported to diminish rapidly on
drying, milling, and storing as well as after mixing with other ingredients to make
poultry feeds (Anthony and Shankaranarayana, 2001).
Lutein is stable in pH range 3 to 9. At extreme pH and in the presence of light,
lutein undergoes isomerization resulting in color loss. Lutein structure consists of
conjugated bonds, which when react with 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, and alcohols. Extensive oxidation results in bleaching of
carotenoid pigments. To minimize color loss, it is safe to pack lutein-containing
products in tin or opaque containers. Enzymes like lipoxygenase hasten oxidative
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Colorant from Marigold-Chemistry 37

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Figure 1. Structures of lutein isomers of marigold.

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38 Sowbhagya, Sampathu, and Krishnamurthy

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. Blanching exhibits an apparent
increase in xanthophylls content due to inactivation of lipoxygenase and also
enhances pigment extraction (Alam et al., 1968).
Marigold carotenoids have potential as a natural food colorant. The status of
marigold, as a source of natural carotenoids, has been reviewd (Verghese, 1998a,
1998b). Stability of xanthophyll pigment extracted from aztec marigold has been
studied wherein oil-in-water emulsions were formulated by dispersing marigold
oleoresin in mesquite or arabic gums. At higher concentration of mesquite gum, the
stability of the color also increased in the emulsion. Color degradation and
coalescence kinetics of aztec marigold oleoresin-in-water emulsions stabilized by
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mesquite or arabic gums also have been studied. The composition of emulsifying
agents and pH have an important role in determining the degree of effectiveness of
the emulsions against color loss and coalescence kinetics. Mesquite gum is reported
to give more stability than gum Arabic (Vernon Carter et al., 1996).

ANALYSIS OF PIGMENTS

Gas chromatographic analysis of methyl esters derived from the hydrolysate of

marigold extract has indicated that xanthoplylls are acylated with palmitic (60.4%),
myristic (22.6%), stearic (14.4%), lauric (92.4%), and oleic (traces) acids (Alam et al.,
1968). By combination of thin-layer chromatography (TLC) and UV spectrometry,
the presence of dipalmitate, dimyristate, and monomyristate have been confirmed
(Gregory et al., 1986). By employing high-performance liquid chromatography (HPLC)
technique, the composition of lutein fatty esters have been reported (Table 2).
HPLC and TLC have been used in the identification and analysis of pigments.
HPLC analysis has been carried out on a C18 column with eluent of medium polarity
such as mixtures of methanol and ethyl acetate in gradient elution. Lutein has been
extracted from fresh petals using acetone:hexane (80:20) solvent mixture. Further
extraction of sample with the same extraction solvent has been carried out, and the
hexane layer was used for quantification. Lutein was crystallized by hot isopropanol
and purified by TLC before HPLC analysis for reference. Silica gel plates were used
and methanol-acetone-petroleum ether (3.5:20:76.5). Lutein concentration was
caluculated by using an E1% value of 2550 in ethanol at 446 nm (Bauernfeind,
1981). It has been concluded from the above work that marigold flower does not
contain free lutein. For analysis, C18 column was used with methanol–ethyl acetate
in gradient elution in two different proportions with UV detetection at 446 nm.
Under the above conditions, lutein along with the esters (viz., lutein dimyristate,
lutein myristate palmitate, lutein dipalmitate, and lutein palmitate-stearate) have
been separated (Gregory et al., 1986) (Fig. 3). The identification of lutein esters was
done based on absorption spectra, TLC separation pattern and HPLC retention
data. The absorption pattern of lutein and lutein esters in different solvents is shown
in Table 4. The structures of xanthophyll esters is shown in Fig. 2.
A method for the separation of free, mono- and diesterified lutein by HPLC has
been reported (Rivas, 1989). This method has the advantage of separation of mixed
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Colorant from Marigold-Chemistry 39

Table 4. Spectral properties of marigold xanthophyll.

Xanthophyll lmax (nm) Solvent

Lutein 474, 446, 420 Hexane

447, 475, 506 Carbon disulphide
Phytoene 296, 285 Hexane
a-Carotene 442, 442, 421 Hexane
b-Carotene 475, 448, 424 Hexane
a-Zeacarotene 449, 424, 399 Hexane
a-Cryptoxanthin 472, 443, 418 Hexane
Antheroxanthin 479, 451, 426 Hexane
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Figure 2. Structures of xanthophyll fatty esters in purified marigold extract.

esters from diesters of xanthophyll and separates free xanthophylls from their fatty
acid esters in a single step. The method consists of using a C18 column with mobile
phase of acetonitrile-methanol-ethyl acetate in gradient elution at 450 nm detection.
Acetone extract of the marigold flower was partitioned with hexane and evaporated,
then taken into ethyl acetate and used for the HPLC analysis. Both saponified and
unsaponified extracts of marigold were analyzed. In total, eight esters of lutein—
namely, lutein monomyristate, monopalmitate, monosterate, dimyristate, myristate
palmitate, diplamitate, palmitate:stearate, and disterate—have been separated
(Fig. 3).
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40 Sowbhagya, Sampathu, and Krishnamurthy

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Figure 3. Typical HPLC chromatograph of marigold flower extract: (1) lutein, (2) lutein
dimyristate, (3) lutein myristate-palmitate, (4) lutein dipalmitate, and (5) lutein palmitate-
stearate. (Adapted from Gregory et al. (1986)).
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Colorant from Marigold-Chemistry 41

Xanthophyll fatty acid esters is marigold flower petals have been identified by
combined gas chromatography-mass spectrometry (GC-MS) and HPLC methods
(Gau et al., 1983). Xanthophyll fatty acid esters were isolated in semipreparative
scale and structure elucidation was carried out by MS. In addition to the major
components viz., xanthophyll dipalmitate, the presence of mixed esters (i.e.,
xanthophyll palmitate sterate, xanthophyll palmitate myristate), which were hither
to unknown, has been established. Liquid–liquid extraction technique using Craig
countercurrent distribution (CCD) is described as a method for the isolation of pure
xanthophyll palmitate in larger quantities. Reversed-phase HPLC with C18 column
was used for the analysis, mobile phase was dichloromethane:acetonitrile (35:65),
and flow rate 15 mL/min detection at 471 nm. The liquid–liquid distribution was
carried out in dimethyl formamide:dichloromethane:hexane (8:2:10 v/v) under argon
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in the absence of light, using a recycling procedure.

A spectrophotometric method for the estimation of xanthophyll content in
marigold oleoresin has been given by AOAC (Helrich, 1990). A mixture of solvents
(viz., acetone þ hexane þ alcohol þ toluene) is reported for the extraction followed
by hot saponification. After saponification, lutein is extracted into the hexane layer
and absorbance of the solution is measured at 474 nm against hexane as reference.
Total xanthophylls ¼ A474  D
W  236
A474 ¼ absorbance at 474 nm D ¼ final dilution
W ¼ sample weight 236 ¼ trans lutein specific absorbtivity for g/L
The correlation of HPLC and AOAC methods to assess the all-trans lutein
content in marigold flowers has been studied (Delgado Vergas and Paredes-Lopez,
1996). Pigments were extracted from marigold meal using an AOAC modified
method (Chen, 1992). HPLC method was established to resolve lutein isomers in
marigold extracts using a triacontyl polymeric surface-C30 column. A regression
equation was established to relate total xanthophylls by AOAC methods with the all-
trans-lutein contents assessed by HPLC. Four major lutein isomers were detected
after saponification of the marigold extract. Total xanthopohylls by this modified
method showed a good correlation with all-trans-lutein content.

PROCESSING

Full-blown marigold flowers having minimum calyx portion are taken for
processing. The availability of flower is seasonal; hence, during peak season the
flowers are collected and stored suitably. The harvested flowers are filled into
chambers (rooms) of about 70 to 80 tons holding capacity having drainage ducts.
The room is constructed in such a way that the three sides are brick walled and the
other side is closed with wooden plank having a discharge chamber to withdraw the
material, as and when required, for further processing. After the material is loaded
into the room, it is compressed and sprayed with lactic bacterial culture, covered
with a layer of lime and covered with black tarpaulin. This will induce lactic acid
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42 Sowbhagya, Sampathu, and Krishnamurthy

fermentation under anaerobic condition. Under this condition, the material can be
stored for 3 to 4 months. By this technique, it is reported that xanthophylls are
stabilized and preserved (Vernon Carter et al., 1996). The extudate water will be
drained from the bottom. The amount of pigment lost in the outgoing liquor is
negligible. The raw material, after withdrawal from the room, is passed through a
dewatering unit and then dried in a drier for 8 to 10 hr under controlled conditions at
a temperature lower than 60
C to 65
C to a moisture level of 8% to 10%. The dried
flowers are powdered (15–20 mesh size) and made into pellets (8  12 mm) after
conditioning of the powder. Pellets are loaded into extractors and extracted using
hexane as the solvent.
In our laboratory, studies on the storage of fresh marigold petals were carried
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out by two techniques of storage: (1) anaerobic storage, and (2) lactic culture method
(unpublished data).

Anaerobic Storage

Petals (1 kg) separated from calyx were mixed with distilled water (200 mL)
and packed aspectically into 10 glass bottles (100 g each), taking care to press
out most of the air, and the lids were secured tightly to prevent external
contamination. The bottles were stored at ambient conditions. Samples were
withdrawn periodically after 12, 18, 35, and 135 days and analyzed for pigment
and also for spoilage by visual observation. No spoilage was observed even after
135 days of storage. The initial pigment in the treated material was 0.25% (as-is
basis) and there was no change in pigment content after 135 days of anaerobic
storage.

Lactic Culture Method

Petals (1 kg) were mixed with an aqueous suspension (200 mL) containing two
types of lactic bacteria culture and aseptically packed into glass bottles (100 g each).
The bottles were stored at ambient conditions, and the samples were withdrawn
periodically after 12, 18, 35, and 135 days and analyzed for pigment content. The
petals were free of spoilage and retained bright orange color, and there was no
pigment loss at the end of 135 days of storage. Both techniques, viz., anaerobic
storage and lactic preservation, showed promise in extending the storage life of
petals beyond 135 days.
Marigold flowers stored by anaerobic and lactic methods were subjected to
mechanical pressing using a hydraulic press before drying to reduce the moisture
content in the petals because this helps to reduce the load on the drier and the drying
time. The drying time for 100 g petals was reduced from 6 hr (control) to 2 hr for
pressed petals.
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Colorant from Marigold-Chemistry 43

Oleoresin Preparation

The dry yield of flowers is about 10%, which contains about 1% to 2%

xanthophyll. Quality of the starting material and conditions of dehydration are two
of the critical factors that influence the retention of xanthophylls. Hexane has been
found to be the best solvent for the extraction of xanthophylls and soxhlet extraction
technique is usually followed and, in some cases, the extract itself is recycled. The
extract (miscella) so obtained is distilled under controlled conditions. Sometimes
extraction is carried out at higher temperatures (40–45
C) to get higher yields of
xanthophylls. For better retention of xanthophylls, extraction temperature needs to
be maintained between 40
C and 45
C and removal of solvent from miscella is
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carried out under vaccum between 50
C and 55
C. The yield of oleoresin is about
8% to 10% with a xanthophyll content of 8 to 12 g per 100 g oleoresin. Sometimes an
antioxidant like ethoxyquin at 0.1% to 0.3% is added to the final product with
stirring at a temperature of less than 45
C for the stabilization of the pigment.
Concentration of lutein fatty acid esters in marigold extracts can be enhanced by
purification steps using solvents like isopropanol followed by precipitation.
Enrichment of xanthophylls can be achieved by phase partition between 70% and
90% of methanol, ethanol, acetone, and hexane. In one of the methods, marigold
extract is subjected to precipitation using isopropanol, which removes 65% of the
lipids in the extract. The precipitated fraction contained 51.3% lutein esters and a
second precipitation from isopropanol-petroleum ether (80:20) resulted in a product
of much higher purity, more than 65% pure. The precipitated lutein esters were
soluble in vegetable oil up to 20% w/w at 60
C (Philip and Berry, 1976). Properties
of purified lutein fatty acid esters are provided in Table 5. Hexane-extracted

Table 5. Properties of purified lutein fatty acid esters.

Property

Yield 21 g/kg dried marigold petals

Lutein ester content 51%
Absorption (carbon disulphide) 473 and 504 nm
Melting range 43–53
C
Solubility in hydrogenated fat at 60
C 20 g/100 g
Solubility in hydrogenated fat at 80
C Miscible
Fatty acid composition (%)
C10:0 2.4
C12:0 17.1
C14:0 30.4
C16:0 15.3
C18:0 5.6
C18:1 12.7
C18:2 4.7
C18:3 0
C20:0 11.8
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44 Sowbhagya, Sampathu, and Krishnamurthy

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Figure 4. HPLC chromatograph of an unsaponified marigold extract: 1, lutein; 2, lutein

monostearate; 3, mono palmitate; 4, monostearate; 5, dimyristate; 6, myristate palmitate; 7,
dipalmitate; 8, palmitate stearate; and 9, distearate. (Adapted from Rivas (1989)).

marigold oleoresin is dark brown in color and highly viscous, with a characteristic
herbal odor. Commercial extracts are valued by their xanthop,hyll and trans lutein
content. It is reported that xanthophyll content in nonsaponified marigold oleoresin
will be 8% minimum and desirable is 9% to 11%, which is preferred in the trade,
although 7.5% minimum is acceptable (Verghese, 1998b).
Application of enzymes for enhanced extraction of xanthophylls from marigold
flowers has been studied (Delgado-Vergas, 1997; Delgado-Vargas and Pardes-Lopez,
1997). Commercial enzyme preparation was used for the pretreatment of fresh
marigold flowers at 0.01% at pH 5 adjusted with citric acid and kept at room
temperature for 120 hr. After enzyme treatment, water-soluble substances were
removed by washing with deionized water. Further extraction was carried by the
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Colorant from Marigold-Chemistry 45

AOAC method by using a mixture of solvents. By enzyme extraction, the yield

of carotenoids increased from 18 to 24.7 g/kg. The commercial enzyme used
was a mixture of cellulase, b-glucanase, and b-glucosidase. Enzyme-mediated
solvent extraction of carotenoids from marigold flower has also been carried out
(Delgado-Vergas and Pardes-Lopez, 2002).

Saponification

Saponification of marigold oleoresin deesterifies the pigment to free xantho-

phylls, and these saponified products find application in poultry feeds to improve the
color of the egg yolk and muscle. Saponification of marigold is carried out by adding
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40% methanolic KOH to the extract and refluxing at 55
C for 20 min. and after
cooling, hexane is added, shaken well, and kept in dark for 2 hr. Xanthophylls are
extracted to the hexane layer, which is used for the estimation of xanthophylls
(Quackenbush, 1973). Most of the commercial saponified extracts contain 1% to 4%
xanthophyll. Saponified oleresin is also modified into dry powder by absorption into
materials like calcium silicate, saturated fatty material, gelatin, gum acacia, and
starch. Some food color preparations containing purified lutein are also available.
Commercial marigold extract is available in different forms.
Several patents examine the extraction and isolation of lutein, zeaxanthin, and
other carotenoids from marigold flowers and oleoresin. According to one of the
patents (Frederick Khachik, 1995), a process for simultaneous extraction,
saponification, and isolation of lutein and zeaxanthin and a mixture of several
carotenoids in high purity from plants without the use of harmful solvents is
described. Lutein crystals containing 5% zeaxanthin were obtained from the dried
petals of marigold flowers (T. erecta), whereas zeaxanthin was isolated and purified
from the berries of Lycium Chinese Mill (LCM). The method employs a
combination of tetrahydrofuran (THF) and alcoholic potassium hydroxide at
room temperature under mild conditions to quantitatively effect the hydrolysis of
lutein and zeaxanthin fatty acid esters in marigold flowers and LCM berries to their
free forms. This method can easily remove the unwanted chlorophylls. After
filtration, the THF and alcohol are evaporated and the residue is washed well with
water and alcohol to remove the THF and the base, and in the case of the green
plants, the chlorophylls and their derivatives as well. The resulting crystals of lutein
and zeaxanthin obtained from their respective sources are approximately 70% pure
and can be further purified to 97% either by recrystallization or by passing their
solution in tetrahydrofuran/water through a silica gel column. As by-products of
these latter purification steps, several rare carotenoids such as anhydrolutein and
alpha cryptoxanthin from marigold and b-cryptoxanthin from LCM berries are
obtained. The pure crystals obtained are claimed to be suitable for human consump-
tion and can be used as a nutritional supplement and as an additive in foods.
According to one of the patents, a process for the isolation and purification of
xanthophylls, particularly lutein from marigold flower petals, zeaxanthin from
wolfberries, or capsanthin and capsorubin from red pepper, is described (Rodney
and David, 1997). The plant extract containing a xanthophyll diester is saponified in
a mixture of propylene glycol and aqueous alkali to obtain xanthophyll crystals
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46 Sowbhagya, Sampathu, and Krishnamurthy

without using organic solvents. The crystals are isolated and purified. The
substantially pure xanthophyll crystals so obtained are claimed to be suitable for
human consumption and also for use as a nutritional supplement and an additive in
foods.
In another patent (Luis, 1999), a method for the preparation of high-purity
trans-xanthophyll ester concentrate is reported. It is claimed in the patent that, by
the method reported, a preparation having trans-xanthophyll ester content four to
nine times higher than the cis-xanthophyll ester content can be obtained. Only the
corollas of marigold flower have been used to get the lutein ester concentrate of high
purity. The structural conformation of the isolated lutein and its esters isolated from
marigold flowers has also been determined (Frederick Khachik and Andrea Steck,
1999).
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A patent is available for the preparation of xanthoplhyll formulations (Philips,

1977). In one of the formulations, 1% solution of lutein fatty acid esters is prepared
by dissolving in hot mono- and diglycerides and homogenized with an equal volume
of 0.5% solution of polysorbate 80. In another formulation, a mixture of hydro-
genated vegetable oil (5 g), mono- and diglycerides (5 g), and lutein fatty acid esters
(200–1000 mg) is dissolved in acetone (100 mL), and the solution is mixed with
ground citrus juice sacs (100 g). The dried product is dispersed in water (2 g/100 mL),
sucrose (13 g/100 mL), pH adjusted to 3 with citric acid and flavored with orange oil
(Grum-Grzhimalo et al., 1983).

FAO Specifications

The Food and Agriculture Organization states that Tagetes extract (a synonym
for xanthophylls) is obtained from hexane extraction of dried petals of T. erecta L.
and subsequent removal of solvent. The content of total coloring matter is calculated
as lutein. According to the Code of Federal Regulations (Marmion, 1985), hexane
extract of marigold flowers petals will conform to the following specifications:

Melting point: 53.5–55
C

Iodine value: 132–145
C
Saponification value: 175–200
Acid value: 0.60–1.20
Titer value: 35.5–37.0
Unsaponiable matter: 23.0–27%
Hexane residue: less than 25 ppm
Tagetes meal and extract has been listed with a color index of 75125, and it is
allowed in chicken feed only to a maximum limit of 1% (Soboleva et al., 1978).

APPLICATIONS OF MARIGOLD COLOR

Dried marigold flowers are used in poultry feed, either in powder from or as
extracts, which imparts a good yellow color for egg yolks and muscle tissue. Lutein
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Colorant from Marigold-Chemistry 47

esters are soluble in vegetable oil to an extent of 20% to 25% (w/w) and can find
application as a food colorant. Marigold color can find application in coloring foods
like edible oils, oleo-margarine, mayonnaise, mustard and other salad dressings,
yogurt, cakes, ice cream, and dairy products. Marigold extract without purification
is not allowed directly in foods. Only the purified extracts with a lutein content of
known concentration and a pure crystalline lutein isolated from marigold flower is
allowed for food use. Recently, the safety aspect of dietary incorporation of lutein
(86% pure) isolated from marigold flowers in a 4-week rat feeding study has been
carried out (Kruger et al., 2002). The bioavailability of lutein and zeaxanthin has
also been studied. The authors have concluded that crystalline lutein is safe and a
generally recognized as safe (GRAS) source of lutein for uses in food.
According to a USSR patent (Zotov et al., 1990), it is reported that 5% aqueous
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alcoholic infusion of marigold flower (4–6%) is used in the preparation of a

nonalcoholic beverage by the name of ‘‘Melpol.’’ Other components include starch
(25–30%), castor sugar (10%), citric acid (0.17–2%), honey (5–7%), and aerated
water.
Petals of marigold are a good source of pigment for avian feed (Alam et al.,
1968; Lettner and Stephan, 1981). The petals were mixed with feed and fed to
chickens at 33, 66, and 99 mg pigment/kg of low-pigment diet. Diet with pigment
content of 33 mg/kg feed produced yolk color acceptable to the consumer. With an
increase in the amount of pigment in the feed, color deposition in the organs
increased, but efficiency of utilization was lowered. Diets containing saponified and
nonsaponified marigold extract were fed to a group of broilers and it was observed
that the saponified marigold extract markedly increased the skin coloration
capability. Yellowness scores of the skin were considerably increased (Flectcher
and Papa, 1986).
The effect of marigold extract in combination with paprika oleoresin on
coloration of egg yolk has been studied. Two commercially available marigold and
paprika oleoresins were used in combination for the feeding trails of hens. Laying
hens were fed with rations containing marigold petal meal, paprika extract, paprika
powder, or corn meal for 20 days. Inclusion of pigment in the diet significantly
increased Roche color fan (RCF) scores in all cases. Paprika supplementation
induced a high degree of red pigmentation, whereas corn gluten and marigold petal
meal induced yellow pigmentation. With equal levels of xanthophyll addition, the
increase in color score was more in the case of paprika than corn and marigold meals
(Guenther et al., 1973). Various combinations of marigold oleoresin (0–60 mg/kg)
and paprika oleoresin (0–12 mg/kg) were used in combinations for feeding laying
hens for 21 days. RCF values were calculated for egg yolks from the various diet
treatments. Color intensity of the yolk was found to increase with increasing dietary
xanthophylls concentration, irrespective of xanthophylls source. Pigment capacity of
paprika oleoresin was greater than that of the marigold. A concentration of 3 mg
xanthophylls from paprika gave the same Roche color value equivalent to 60 mg
xanthophyll from marigold. No interaction of paprika and marigold xanthophylls
has been observed (Fletcher and Halloran, 1981, 1983; Nakajima et al., 1994).
Addition of marigold color (dried petals of marigold) to butter creams has been
studied, and it was found to increase the quality and shelf life of butter creams.
Addition of marigold petals or dried flowers of marigold in butter in confectionery
 ORDER REPRINTS

48 Sowbhagya, Sampathu, and Krishnamurthy

creams prepared from 1:1 mixture of butter:sugar, and in concentrated milk (40%),
was carried out. Products containing 0.4% marigold powder and controls were
stored at 0
C for 7 days, and it was found that the addition of marigold color
imparted an intense yellow color and intensified the flavor and aroma of the creams.
After the storage period, color and flavor of treated samples decreased only slightly
without any off-flavor development, whereas the control formulations acquired a
pronounced tallowy odor. The study recommended the addition of marigold color to
butter creams for the improvement of quality (Soboleva et al., 1978).

CONCLUSION
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Although marigold colorant offers a strong intense orange to yellow color, a

good substitute for yellow/orange synthetic colorant, no toxicity data are available in
the literature on marigold extract either partially purified or the total extract, which
makes it unusable in food. Marigold has a high tinctorial power and good heat and
light stability. Compared with other natural sources of yellow and orange color, like
turmeric, chill, and saffron, marigold is a cheaper source and easily available.
Marigold flower can be a cheaper source as a starting material for the isolation of
lutein, the much-valued natural pigment that can also serve as a nutraceutical.
However storage of marigold flowers in seasonal times is very important. A suitable
technique for storage enhances the stability of the pigment in the flower. Anaerobic
and lactic acid treatments of fresh flowers is promising in terms of pigment stability.
It is widely known that carotenoids are beneficial for human health. However,
the biological functions of many individual carotenoids like zeaxanthin, crypto-
xanthin, antheraxanthlin, and neoxanthin, which are present to a large extent in
marigold flowers, is not known. For example, the dipalmitate ester of lutein is used
as a major component of eye ointment for its antioxidant property.
Lutein is a natural pigment that can give a good orange shade to foods.
Information on isolation of lutein from marigold extract, its stability on isolation,
and application in foods and stability is not easily available, although a few patents
can be found on extraction and isolation of lutein. Detailed work in these directions
will be necessary, as marigold is abundantly available.
Being rich in source of lutein and other carotenoids, marigold flowers make an
excellent source of valuable nutraceuticals. With the application of biotechnology,
newer varieties of marigold with higher lutein content can be grown. As it stands,
only purified lutein can be used in food applications for human consumption.
Toxicological studies of partially purified marigold extracts are needed so that they
can be used as the source of natural food colorant. With more toxicological studies
showing its safety, marigold flowers can be good source of natural orange colorant
in food applications.

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Colorant from Marigold-Chemistry 49

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