Phytochemical

Analysis
46
P. GUINOT
ET AL.
Phytochem. Anal. 19: 4651 (2008)
Published online 24 July 2007 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/pca.1014

Phytochemical
Analysis

Primary Flavonoids in Marigold Dye: Extraction, Structure

and Involvement in the Dyeing Process
PAULINE GUINOT,1 ANNICK GARGADENNEC,1 GILLES VALETTE,2 ALAIN FRUCHIER3 and CLAUDE ANDARY1*
1

Laboratoire de Botanique, Phytochimie et Mycologie, Facult de Pharmacie, UMR 5175 (CEFE), 15 avenue Charles Flahault, 34093 Montpellier Cedex
5, France
2
Laboratoire de Mesures Physiques, Universit de Montpellier 2, cc 010, place Eugne Bataillon, 34095 Montpellier Cedex 5, France
3
Ecole Nationale Suprieure de Chimie, UMR 5076, 8 rue de lEcole Normale, 34 296 Montpellier Cedex 5, France
Received 21 January 2007; Revised 23 May 2007; Accepted 29 March 2007

Abstract: Flavonoids extracted from marigold flowers were investigated for their dyeing potential. Patulitrin (1) and patuletin (2)
were isolated and their structures established using NMR and HPLC-MS. These compounds were identified as the main flavonoids
present in the dyeing bath. Following the dyeing process, it was demonstrated that aglycone 2 bound more strongly to wool fibres
than its glucoside 1. Moreover, analysis focused on 1 and 2 dynamics during plant growth revealed that these components were
only found in flowers during and after flowering. The influence of growing location was also investigated and it appeared that
cultivation under Mediterranean conditions enhanced biosynthesis of 1 and 2. Finally, several solvents were tested for their potential to extract the flavonoids: the use of a waterethanol mixture gave a high extraction efficiency and allowed selective extraction of
1 and 2. The implications of these results are discussed in relation to the development of marigold as a potential dyeing plant.
Copyright 2007 John Wiley & Sons, Ltd.
Keywords: Tagetes patula; HPLC-MS; NMR; flavonoid extraction; dyeing process; patulitrin; patuletin.

INTRODUCTION
Natural dyes have been extensively used since ancient
times as textile colouring matters to dye fibres. Advances
in chemical techniques rendered possible the production of synthetic dyes at relatively low cost and dyes
produced from natural sources were less competitive
and were progressively replaced by synthetic dyes.
Nowadays, the use and production of natural dyes is
becoming more popular owing to the growing awareness
of environmental problems coupled with the toxicity
associated with synthetic dyes (Anliker et al., 1988).
As a result, renewable resources are now being reinvestigated as alternative raw materials (Angelini et al.,
1997; Cristea et al., 2003; Vankar et al., 2007). Furthermore, natural dyes can exhibit better biodegradability and represent a more environmentally friendly
alternative to synthetic dyes (Frischer and Garcia, 2005).
Some of them have also been reported to possess antiUV and anti-microbial properties (Singh et al., 2005).
In the continuation of our previous work on dyeing
plants (Guinot et al., 2006), we have focused on marigold, Tagetes patula L. in the present study. Using this
dye, the resulting shades obtained on both cotton and

* Correspondence to: C. Andary, Laboratoire de Botanique,

Phytochimie et Mycologie, Facult de Pharmacie, UMR 5175 (CEFE),
15 avenue Charles Flahault, 34 093 Montpellier Cedex 5, France.
E-mail: candary@ww3.pharma.univ-montp1.fr
Contract/grant sponsor: The FEDER Program.
Copyright 2007 John Wiley & Sons, Ltd.

wool yarns were quite dark and saturated, characteristics that are particularly valued in dye formulation.
Moreover, light fastness was satisfactory while resistance to water was excellent. Furthermore, marigold is a
raw material of particular interest, not just in relation
to dyeing but also with regard to its functional properties. The carotenoid lutein, which is found in marigold,
is currently used as food colorant approved by the EU
(E161b). Marigold extracts are also approved as feeding
additives to improve the pigmentation of poultry skin
and eggs. It has also been suggested that carotenoids,
such as lutein, may protect against certain eye diseases (Johnson et al., 2000; Krinsky and Johnson,
2005). Moreover, the essential oils from flowers are
reported to have larvicidal (Dharmagadda et al., 2005),
antifungal (Mares et al., 2004; Romagnoli et al., 2005)
and antimicrobial activities (Piccaglia et al., 1996). As
a result, marigold is a plant material of particular
interest in relation to its present uses and biological
properties. Since flavonoids, carotenoids and essential
oils are not extracted under the same conditions
(Bruneton, 1999), the production of dyeing extracts as
by-products of the exploitation of marigold for other
uses is possible, as has already been suggested in previous works on other raw materials (Betchold et al.,
2006; Guinot et al., 2007).
Previous phytochemical studies of marigold generally
focussed on the essential oils from flowers (Marotti
et al., 2004; Vidya Sagar et al., 2005) and on the carotenoid (Piccaglia et al., 1998) or monoterpene content
Phytochem. Anal. 19: 4651 (2008)
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PRIMARY FLAVONOIDS IN MARIGOLD DYE

47

Table 1 Raw materials harvested during plant growth

Growing stage
Flower buds formation
Flowering
Flower fading
Fruit formation

Figure 1 Chemical structure of patulitrin (1), patuletin (2)

and quercetagetin 7-O-glucoside (3).

(Garg et al., 1990), but there are few reports on the

phenolic composition of this plant. Flavonoids, such as
kaempferol or quercetin-like structures, have already
been reported (Ivencheva and Zdravkova, 1993). Furthermore, Tarpo (1967, 1968, 1969) and Bhardwaj et al.
(1980), reported the presence of patuletin 7-O-glucoside
(patulitrin) and of patuletin (Fig. 1), quercetagetin,
quercetagetin 7-O-glucoside (Fig. 1) and luteolin, but
no structural evidence was provided to confirm the
structures of patulitrin (1) and patuletin (2).
In order to obtain useful information for the development of a new natural dye from marigold, this paper
presents conclusive structural analysis of the main
flavonoids present in a traditional marigold dyeing bath
and that are involved in the dyeing process of wool (a
protein-based fibre chosen as a being one of the most
common yarns used with natural dyes). A quantification method was developed to analyse 1 and 2 in various samples. Different extraction methods of the dye
components were compared in order to identify the
most efficient. Lastly, the evolution of flavonoids during
plant growth and the influence of growing location on
the flavonoids contents in the plant were evaluated.

EXPERIMENTAL
Chemicals and reference compounds. Acetonitrile
(HPLC-grade) and absolute ethanol were obtained
from VWR International (Fontenay-sous-Bois, France).
Trifluoroacetic acid (TFA) was obtained from Fluka
(Sigma-Aldrich, Saint Quentin Fallavier, France).
Purified distilled water was used for all analysis and
extraction. All HPLC solvents were filtered prior to use
by vacuum filtration over a 0.45 m filter. Rutin and
glucose standards were purchased from Fluka and
Carlo Erba (Val de Reuil, France) respectively.
Plant materials. Marigold flowers were collected in Le
Jardin Conservatoire des Plantes Tinctoriales (Lauris,
France) during flowering. Plant material was authenticated by Professor Claude Andary (Laboratoire de
Botanique from Faculty of Pharmacy, University of
Copyright 2007 John Wiley & Sons, Ltd.

Harvested plant material

Leaves
Flowersleaves with stems
Flowersleaves with stems
Fruits with seedsleaves with stems

Montpellier) and a voucher specimen deposited in the

herbarium of the same Laboratory with the reference
number: MPU 113-05. In order to investigate the
evolution of flavonoids during plant growth and the
influence of growing location on flavonoid content,
marigold plantations were established from the same
batch of original seed material simultaneously in Le
Jardin Conservatoire des Plantes Tinctoriales (Lauris,
South of France, Mediterranean climate) and in Le
Jardin Botanique de la Facult de Pharmacie de Lille
(Lille, North of France, temperate climate). Samplings
were carried out at different growth stages (Table 1).
Plants were directly air-dried after harvesting and
then transported to the laboratory and powdered in a
domestic grinder before analysis.

Sample preparation. Crude extracts were prepared as

described in traditional recipes for the preparation of
dyeing baths, i.e. dried ground flowers were extracted
by decoction for 10 min in distilled water (1:100, w/v).
To optimise extraction yield, a number of alternative
procedures were investigated. Extracts of the dried
ground flowers were obtained by maceration (1:100,
w/v) at 60C for 30 min and 60 min under mechanical
stirring (180 rpm). The solvents used were ethanol:
water mixtures of various compositions, i.e. 2:8, 3:7,
5:5, v/v. The extracts used to follow flavonoid evolution
during plant growth and the influence of growing location on flavonoid content were prepared by maceration
with ethanol:water (3:7, v/v) for 30 min at 60C under
mechanical stirring (180 rpm). All the resulting suspensions were filtered through gauze and the filtrates were
made up to their initial volume with the appropriate
solvent. Two millilitres of filtrate were centrifuged for
10 min (7176 g) before analysis. All extractions were
carried out in triplicate.
Dyeing process. Woven wool (Plantes et Couleurs,
Lauris, France) was mordanted as previously described
by Guinot et al. (2006). Then, the dried fabric was
dipped into a dye bath prepared as described above
following the traditional recipe and using a plant to
solvent ratio of 5:100 (w/v). The ratio between dried
plant and textile to dye (wdried plant/wtextile to dye) was 1.5.
The temperature was raised to boiling point under
magnetic stirring and boiling was maintained for a
further 3 min. The textile was then cooled in the dyeing
bath for 60 min at room temperature before being
Phytochem. Anal. 19: 4651 (2008)
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48

P. GUINOT ET AL.

washed three times in 150 mL of distilled water. Dyed

samples were air-dried at room temperature. Two
millilitres of the dyeing bath were analysed before and
after dyeing by HPLC-PAD analysis. Aliquots (2 mL) of
each rinsing water were also analysed to determine the
quantity of dye released during the rinsing process,
and thus to calculate accurately the amount of dye
permanently fixed on the fibre. Dyeings and related
analysis were carried out in triplicate.

Isolation of the main flavonoids. Dried ground flowers

(50 g) were extracted twice in 800 mL of ethanol:water
(8:2, v/v) by ultrasonication for 20 min at 24 kHz. After
filtration through filter paper, the solution was concentrated to 20 mL under vacuum. The resulting crude
extract was purified on a cellulose stationary phase
(Avicel, Merck, VWR International, Fontenay-sous-Bois,
France) using medium pressure liquid chromatography
(MPLC) (45 bars). Elution was carried out by successively percolating water, water:methanol (2:8 and 5:5,
v/v) and pure methanol through the column. The collected fractions were monitored by TLC. The main fractions containing molecules 1 and 2 were purified by
semi-preparative HPLC with a Waters (Saint Quentinen-Yvelines, France) Sunfire C18 column (250 10 mm
i.d.) using the gradient scheme previously described
(see above) at 5 mL/min. Compounds 1 (5 mg) and 2
(5 mg) were isolated in a good state of purity (>95% by
NMR). Compound 1 (4 mg) was dissolved in ethanol:
water (5:2, v/v) and an equal volume of 3 M hydrochloric acid was added to hydrolyse the glycoside. The
solution was placed in an oven for 10 h at 80C and
extraction of the aglycone form was carried out by
partitioning the acidic solution four times with ethyl
acetate (1:1, v/v). The combined organic extracts were
dried over anhydrous sodium sulphate and evaporated
to dryness. The aqueous extracts were filtered prior to
evaporation. The organic fractions were dissolved in
ethanol:water (1:1, v/v) (solution A1) and aqueous
fractions were dissolved in ethanol:water (2:8, v/v)
(solution A2).
TLC analysis. Fractions (5 L) were collected from the
MPLC purification step, spotted onto cellulose TLC plates
(Merck, ref. 5552) and developed with dichloromethane:
acetic acid:water (50:45:15, v/v/v) as mobile phase. A1
solution and 2 were also spotted onto cellulose plate
and developed using the same mobile phase. Spots were
detected by spraying with Neus reagent (Neu, 1956)
and the plates were observed under UV light at 366 nm.
Aliquots (5 L) of A2 solution were spotted onto a silica
gel TLC plate in the presence of glucose standard
(1:1000, w/v in methanol:water (1:1, v/v) and developed
in ethyl acetate:isopropanol:water (2:7:7, v/v/v). After
spraying with a solution of triphenyltetrazonium basic
in methanol (4:100, w/v), the plate was observed under
visible light after heating for 15 min at 100C.
Copyright 2007 John Wiley & Sons, Ltd.

HPLC analysis. HPLC-PAD analyses were performed

using a TSP SpectraSysten from ThermoElectron S.A.
(Courtaboeuf, France). Separation was carried out at
25C on a Waters SunFire C18 column (250 4.6 mm i.d.)
protected by a C18 pre-column. Twenty microlitres of
extract were injected and eluted at 1 mL/min with
acetronitrile (solvent A) and water:trifluoroacetic acid
(99.9:0.01, v/v) (solvent B). A linear gradient was applied
as follows: 025 min: 20% A to 40% A in B; 2529 min,
40100% A; 2937 min, isocratic elution with 100% A.
Peaks were detected by UV absorbance at 360 nm and
quantification was performed by interpolation on an
external calibration curve using rutin standard solutions.
All data were analysed using a ChromQuest software
system. Analyses were carried out in triplicate.
NMR and HPLC-MS analysis. 1H- and 13C NMR spectra of
sample solutions in CD3OD were recorded at 400.13
and 100.61 MHz, respectively, with a Bruker DRX-400
spectrometer (Bruker-Biospin, Wissembourg, France).
Chemical shifts are reported in ppm/TMS and coupling constants are expressed in Hz. Standard Bruker
sequences were used for correlation spectra (COSYGP,
HMQCGP and HMBCGP). 1H- and 13C NMR data of
flavonoid 1 are given below in which proton and carbon
chemical shifts are referred to the solvent, i.e. 3.300 ppm
for CHD2 and 47.84 ppm for CD3 (Hoffman, 2003) 1H
(CD3OD): 3.901 (OCH3-6), 6.917 (H-8), 7.792 (d 1.5 Hz,
H-2), 6.906 (d 8.3 Hz, H-5), 7.701 (dd 8.3 and 1.5 Hz,
H-6), 5.127 (d 7.5 Hz, H-1), 3.589 (H-2), 3.536 (H-3),
3.433 (H-4), 3.571 (H-5), 3.739 (dd 5.9 and 12.1 Hz,
H6a), 3.967 (dd 1.8 & 12.1 Hz, H6b). The signals of
H-2, H-3 and H-5 are not first-order. The seven-spin
system was analysed with the gNMR program to obtain
the three coupling constants: J2,3 = J3,4 = J4,5 = 9.1 Hz.
13
C (CD3OD): 147.63 (C-2), 136.04 (C-3), 176.23 (C-4),
151.70 (C-5 or C-9), 151.77 (C-9 or C-5), 131.95 (C-6),
60.11 (CH3-6), 156.26 (C-7), 93.98 (C-8), 122.54 (C-1),
114.77 (C-2), 144.85 (C-3), 147.63 (C-4), 114.82 (C-5),
120.52 (C-6), 100.61 (C-1), 73.35 (C-2), 76.59 (C-3),
69.89 (C-4), 77.10 (C-5), 61.13 (C-6).
HPLC-MS analyses were carried out on extracts at
room temperature using a Waters Symmetry C18 column
(250 4.6 mm i.d.) with a Waters 2790 Alliance HPLC
system coupled to a Micromass (Manchester, UK) QTof-1 mass spectrometer employing the same mobile
phase and gradient as previously described for HPLCPAD analysis. Peaks were detected with mass spectrometry after electrospray ionisation in the positive ion
mode (heated capillary temperature, 150C; capillary
voltage, 3000 V; cone voltage, 20 and 40 V; desolvation temperature, 260C). Data were processed using a
MassLynx software system.
Colour measurements. Colorimetric data of dyed materials were obtained using a Mercury 2000 Datacolor
spectrophotometer (3C Conseil, Montreuil, France).
Phytochem. Anal. 19: 4651 (2008)
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PRIMARY FLAVONOIDS IN MARIGOLD DYE

49

Figure 2 HPLC chromatogram (360 nm) of dyeing bath obtained from traditional decoction. (1) Patulitrin, (2) patuletin, (3)
quercetagetin 7-O-glucoside (tentative), (46) unknown minor flavonoids.

CIELab values were obtained from CIE D65/10

illuminant-observer reference conditions with an ultrasmall aperture (6 mm). A multi-measurements method
was applied and analyses were carried out in triplicate.

RESULTS AND DISCUSSION

Dyeing bath characterisation and dyeing
mechanism
Figure 2 shows a typical HPLC-PAD chromatogram
obtained by analysis of the dyeing bath before the
dyeing process. Two major peaks were observed at
Rt = 9.00 min (compound 1) and at Rt = 20.48 min
(compound 2). On-line monitoring of the UV/visible
spectrum showed maximum absorbances that correlated
well with the spectral data of patulitrin (1) and patuletin
(2) as described by Mabry et al. (1970): max MeOH = 258,
272sh, 293sh, 371 nm, and max MeOH = 259, 273sh, 338sh,
373 nm respectively. The same extract analysed by
HPLC-MS, gave for 1 a molecular ion [M + H]+ at m/z
495 and a fragment ion from secondary self-fragmentation
at m/z 333. Compound 2 gave a [M + H]+ fragment at
m/z 333 without further self fragmentation. Following
hydrolysis of 1, the Rf values for glucose and solution
A2 were similar and suggested the presence of glucose
in the structure. In addition, the Rf values for solution
A1 and 2 also resembled one another, suggesting a
similar aglycone structure in both 1 and 2.
1
H and 13C NMR spectra of 1 were consistent with
the structure of patulitrin already described by ValantVetschera et al. (2003) in another plant. As compound
1 could be identified as patuletin-7-O-glucoside (patulitrin), compound 2 should be patuletin.
The dyeing bath was also characterised by the presence of minor compounds at Rt = 6.43 min (3), Rt =
11.50 min (4), Rt = 12.21 min (5) and Rt = 13.16 min
Copyright 2007 John Wiley & Sons, Ltd.

(6). HPLC-MS analysis of 3 gave a molecular ion at

[M + H]+ at m/z 481 and a fragment ion from secondary
self-fragmentation at m/z 319, suggesting a possible
glycoside structure. The UV spectrum of 3 presented
typical flavonoid absorbance maxima at max = 258,
278sh, 358 nm, which suggested a quercetagetin-like
structure. Both theoretical UV data and molecular
mass (M = 318 g/mol) were in accordance with the
experimental data. Moreover, quercetagetin and quercetagetin 7-O-glucoside have been previously described
in marigold (Bhardwaj et al., 1980). Unfortunately, due
to the unavailability of appropriate standard, it was
not possible to verify our hypothesis. Compounds 4, 5
and 6 gave the following UV data: max = 252, 256sh,
339 nm, max = 257, 265sh, 359 nm, and max = 252,
272sh, 365 nm, respectively, suggesting flavonoid-like
structures. The HPLC-MS data for the latter three compounds were too weak to be interpretable.
Furthermore, as shown in Fig. 3, the main flavonoids,
1 and 2, are clearly involved in the dyeing mechanism
in particular aglycone 2, which exhibited a higher level
of binding (87%) than its corresponding glucoside 1
(41%) or another possible glycoside 3 (41%). Flavonoids
4 and 6 presented a binding level of 46% while the
binding of 5 approached that of 2 (80%). The value
obtained for the binding 2 could be moderated because
of a possible hydrolysis of glycoside derivatives during the
dyeing process. To verify this hydrolysis, a blank step
(simulating a dyeing without textile) should be carried
out. However, during the extraction of the flavonoids
when the heating time was increased from 30 to 60 min,
the amount of 1 remained nearly the same (Fig. 4)
suggesting an absence or a negligible hydrolysis.
Nevertheless, even if hydrolysis happened, the fact that
aglycone seemed to bind more than glucoside is still
correct, which is of importance from a dyeing point of
view. In fact, findings of 1 and 2 binding on fibres give
for the first time an indication about the compounds
Phytochem. Anal. 19: 4651 (2008)
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50

P. GUINOT ET AL.

Figure 3 Evolution of dyeing bath flavonoids during dyeing process: ( ) mg flavonoid in dyeing bath before dyeing; ( ) mg
flavonoid in dyeing bath after dyeing; ( ) mg flavonoid in rinsing water; (%) percentage of binding flavonoids to fibres during dyeing
process. (1) patulitrin; (2) patuletin; (3) quercetagetin 7-O-glucoside, (46) unknown minor flavonoids.

Table 2 Effect of location and growing conditions on flower flavonoid contents

Percentage of patulitrin (1) in flowers

Percentage of patuletin (2) in flowers

3.2 0.2
1.7 0.27

5.5 0.2
1.5 0.2

Mediterranean conditions (Lauris)

Temperate conditions (Lille)

Figure 4 Influence of different solvents and times on the

yield of extraction of flavonoids: ( ) percentage of patulitrin
(1); ( ) percentage of patuletin (2).

involved in the dyeing process on wool suggesting a

greater involvement of the flavonoid aglycones than their
corresponding glucosides. Similar essays carried out with
a weld (Reseda luteola) dyeing bath supported that
suggestion (unpublished results). Moreover, the dyeing
potential of marigold extracts should not be reduced to
one dyeing molecule but should be attributed to a group
of molecules involved in the dyeing process. Marigold
dyeing extract gave a quite dark yellow shade on wool
characterised by the following CIELab parameters: L* =
55.22 0.36; C* = 46.02 0.65; h = 91.06 0.20.

the presence of these two compounds, although some

hydroxycinnamic acids (max = 330 nm) and other minor
flavonoids (max = 360 nm) were detected in trace quantities. This result was in accordance with the traditional use of marigold: only flowers are quoted as dyeing
material (Cardon and du Chatenet, 1990). Furthermore, the level of both 1 and 2 seemed to be influenced
by growing conditions as shown in Table 2. It appears
that, under a Mediterranean climate (Lauris), the concentration of flavonoids, and in particular compound 2,
is higher than under temperate climate conditions
(Lille). This finding may be attributed to one of the fundamental purposes of flavonoid biosynthesis in plants:
phenolic compounds, and in particular flavonoids,
have been shown to provide plants with protection
against UV radiation (Caldwell et al., 1983; Smith and
Markham, 1998). Thus, a warmer and sunnier climate
may be cause of the higher accumulation of flavonoids
due to greater UV exposure. Similar results were obtained in assays carried out with other dyeing plants
(Serratula tinctoria and Artemisia vulgaris) (unpublished data). Nevertheless, more extensive studies are
needed in order for conclusions about the influences of
these parameters to be drawn.

Influence of extraction methods on flavonoid yields

Evolution of flavonoids during plant growth and
influence of growing location
Firstly, compounds 1 and 2 were only found in flowers
(Table 1). In fact, analysis of the leaves did not show
Copyright 2007 John Wiley & Sons, Ltd.

Figure 4 shows the extraction yield for each of the

solvents investigated. It was clearly observed that water:
ethanol solutions increased flavonoid extraction when
compared with traditional decoction methods. The
quantity of 1 increased in the extract with increasing
Phytochem. Anal. 19: 4651 (2008)
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PRIMARY FLAVONOIDS IN MARIGOLD DYE

amounts of ethanol in the solvent, while the amount of

the aglycone 2 decreased under the same conditions.
Taking into account that 2 seemed to bind more
strongly to wool, ethanol:water (2:8, v/v) mixture could
be the most suitable choice for dye extraction. Moreover, increasing extraction time did not appear to noticeably increase the amount of extracted flavonoids
except for the case of patuletin (2) when ethanol:water
(2:8, v/v) was used (Fig. 4). These results, combined
with both flavonoid evolution during plant growth and
influence of climate conditions on their biosynthesis,
are of importance for the development of new natural
colouring matters from plants such as marigold.

Acknowledgement
The authors would like to gratefully thank Aline Rog
(Laboratoire de Botanique, Facult de Pharmacie de
Lille) and Florent Valentin (Jardin Conservatoire des
Plantes Tinctoriales de Lauris) for growing and harvesting the plant material. The FEDER Program is thanked
for financial support.

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Phytochem. Anal. 19: 4651 (2008)

DOI: 10.1002.pca