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International Journal of Natural Products Research
Universal Research Publications. All rights reserved
ISSN: 2249-0353
Original Article
Isolation, application and biochemical characterization of colour component from
Tecoma stans: A new cost effective and eco-friendly source of natural dye
Arunkumar, P* and A.Yogamoorthi**
*Ph.D.,scholar, ** Associate Professor, Department of Ecology & Environmental Sciences, Pondicherry University
Pondicherry
Received 02 January 2014; accepted 14 January 2014
Abstract
Natural dyes have gained importance due to the growing environmental awareness and implementation of stringent
regulations in production and use of synthetic dyes. This scenario necessitated the present investigation on bio-dyes. The
study revealed three important features viz. flowers of Tecoma stans possesses fast colour yellow colorant -a carotenoid
group of pigments – Zeaxanthin., secondly during dyeing process, pre-mordanting with stannous chloride has yielded
better color fastness., fluorescence decay profile of zeaxanthin in Dichloro methane (DCM) exhibited Excitation (λ) at 420
nm and emission (λ) at 650 nm which strongly support the self quenching property of zeaxanthin. By virtue of such
interesting property, flowers of Tecoma stans is could be a potential source of yellow colourant with striking spectroscopic
characteristics.
© 2014 Universal Research Publications. All rights reserved
Passwords: Tecoma stans-biodye-yellow colourant- dyeing-zeaxanthin-spectral studies
Introduction
Natural dyes are now-a-dyes important not only for textile
industries but also for cosmetics, food and pharmaceutics.
Natural dyes has gained importance due to the growing
environmental awareness and due to the result of stringent
environmental standards imposed by many countries in
response to toxic and overall water quality issues due to
effluents associated with synthetic dyes. Under such
situation natural dyes derived from plants have gained
economic advantage over synthetic dyes because of their
non-toxic, non-carcinogenic and biodegradable nature
(Padhy and Rathi; 1990, Garz et al; 1991, Eom et al; 2001,
Gupta et al;2001, Tiwari & Vankar; 2006). Extensive
works on search for bio-dye has been carried on production
and application (Kamal et al; 2005, Lec et al; 2007,
Bechtold et al; 2007, Vankar & Rakhi; 2009, Shaukat et al;
2009). Natural colorants has been extracted from leaves of
Eucalyptus sp., Cassia tora seeds, Grewia optiva by using
aqueous medium under varying condition and their shades
are imparted in silk,cotton and jute fabrics by Dayal and
Dobhal(2001) and Saxena et al., (2001). Work has also
been carried out to prepare eco-friendly natural dyes from
different parts of Gulmohur flower (Purohit et al., 2007);
from flowers of the Tabebuia argentea plant found to
possess acceptable dyeing performance attributes on silk
yarn (Konaghatta Vinod et al., 2010); Althaea rosea,
Bougainvillea glabra ,Butea monosperma, Caranthus
9
tinctorus, Chukrasia tabularis, Commenlia benghalensis by
Fursule, (2004); in Crocus sativus and Desmodium
multiflorum by Siva, (2007). Coloured components were
extracted from three varities of mariegold flower in
aqueous medium in different proportions (Sarkar et
al,2005) from the coffee seed and its application in silk and
cotton (Teli&Paul,2006), and in African marigold (Tagetes
erecta), by Joshi , (2008). Extensive literature survey
confounded that the flower of Tecoma stans has not been
examined so far and hence present study has been
undertaken to Isolate, apply and biochemically characterise
colour component from Tecoma stans: A new cost effective
and eco-friendly source of natural dye.
2. Materials and methods:
2.1. Preparation of Aqueous extract of flowers of
Tecoma stans
Tecoma stans, a native flower of United states Virgin
islands, is a flowering perennial shrub of Bignonaceae
family. It is an invasive shrubs which spreads rapidly
through cross pollination. In Indian sub continent, it is
grown as live hedge and amenity tree by virtue of its many
yellow trumpet shaped flowers. Locally it is known as
ponnarali or nagashenbagam. The flowers were collected
from trees growing inside Pondicherry university campus.
The flowers were shade-dried in trays, in thin layers,
immediately after picking. Fresh flowers of Tecoma stans
were collected, washed and shade dried. Further it is
International Journal of Natural Products Research 2014; 4(1): 9-11
crushed and immersed in distilled water and allowed to boil
in a beaker kept over water bath for quick extraction for 2
hours at 80 °C. Most of the colorant material was extracted
from flowers by the end of 2 hours. The extract is kept
overnight. Then solution was filtered and the filtration is
used for dyeing cotton material following the traditional
method adopted by Samanta and Agarwal, (2009). 4 types
of mordants viz. alum, ferrous sulphate ,copper sulphate,
stannous chloride to find out most suitable mordant for the
type of source material (flowers) as efficiency of mordant
varies with the type of plant material/bio-dye.
2.2 Dyeing with cotton cloth:
Four pieces of desized cotton cloth were washed in a
solution containing 4% sodium carbonate at 90 °C, keeping
the material to liquor ratio at 1:40. The scoured material
was thoroughly washed with tap water and dried at room
temperature. The scoured material was soaked in clean
water for 15 min prior to dyeing or mordanting. Accurately
weighed cotton cloth were treated with 4 mordants viz.
alum, ferrous sulphate ,copper sulphate, stannous chloride
for Tecoma stans flower extract. The mordant 2% was
dissolved in water to make a liquor ratio 1:20. The wetted
sample was kept into the mordant solution and heated to 70
°C and maintained at the same temperature for 30 minutes
(Dayal et al, 2006). After moderating the cotton samples
were dyed with dye extract, keeping M:L ratio as 1:20 and
temperature of the dye bath was maintained at 70 °C for 45
minutes (Samanta et al., 2003). After dyeing, the dyed
material was left in the dye bath itself over night. The dyed
cloth is taken out of the bath next day; washed well in tape
water and squeezed out. After 3 such washing, the dyed
cotton pieces are dried in open air. To assesses the cotton
fastness of the dye, the dyed cotton cloths is soaked and
washed in 0.2% soap water and squeezed and dried at open
air. The brightness of the colour of the dyed cloth was
compared with the colour of the pre washing visually.
2.4 Isolation of dye compound for spectral studies.
Tecoma stans flowers from the plant and air dried at room
temperature. The dried flowers were powdered and then the
powdered sample was subjected to soxhlet extraction using
methanol for three hours. The methanolic extract was
obtained after the evaporation of solvent under reduced
pressure. The obtained extract was partitioned using water
and ethyl acetate. The ethyl acetate fraction (5g) was
subjected to silica gel column chromatography eluting with
hexane. The eluted fraction is used for spectral studies.
2.5 Biochemical characterisation
The fraction obtained was analyzed using UV-visible
spectrophotometer in the visible region of 400-800 nm with
different solvents such as hexane, chloroform, methanol
and ethyl acetate and the absorbance value at a wavelength
of maximum or peak absorbance. The fluorescence
properties and life time studies were done using
fluorescence spectrophotometer. The structure of the
obtained fraction was also determined using NMR and
FTIR spectrum. 1H(400 MHz),13C(100 MHZ) were obtained
with CD3OD with FTNMR spectrometer model Avance-II
(Bruker). UV absorption spectra of the compound (dye) in
various solvents such as hexane, chloroform, methanol and
ethyl acetate were recorded using UV spectrometer. FTIR
10
spectrum was recorded using ThermoNicolet FTIR model
:(6700). The fluorescence emission and excitation spectra
were taken on JY Fluorolog-FL3-11 spectrofluorometer with resolution of 0.2nm.
Results and discussion:
The dye bath which is pre mordanted with stannous
chloride and alum resulted better result than post
mordanting. Hence pre-mordanting technique is found
better for this dye extract. Among the four mordants,
stannous chloride yielded better shade and hue (Fig.1).
Fig.1. Cotton cloth dyed with extract of Tecoma stans
using stannous chloride as mordant
Thin Layer Chromatography
TLC of Tecoma stans fraction in methanol extract was
carried out. Better elution is reported when 2.5 ml hexane
with 2.5 ml chloroform solvent mixture were used and the
rf value of the colourant was noted as 0.56 (fig.2) which
almost similar to the r value obtained by Davies et al,
(1970) for Zeaxanthin i.e.0.53
Fig.2. Chromogram of extract
Absorption spectroscopy:
The electronic spectroscopy results of the fraction (a,b,c &
d of fig.3) display two strong absorption bands at 240 nm
and 406 nm namely π- π* . Similar spectra of excited state
of Zeaxanthin were also obtained by Billsten et al., (2005)
and Justyna Milanowska & Wiesław Gruzecki,(2005) for
zeaxanthain f
Infrared spectrum
The FT-IR spectrum of fraction shows the stretching
vibrations of 2923 and 2852 cm-1 whose origins are due to
International Journal of Natural Products Research 2014; 4(1): 9-11
the stretching vibration of aliphatic CH2 groups. Newly
observed broad peak at 3452 indicates aliphatic -OH group
(fig.4). Similar, FTIR spectroscopy results were matched
with the spectrum obtained for flowers of Radermachera
ignea by Duangnapa Sompong and Pichaya Trakanrungroj
(2010).
Fig.3.UV Visible spectroscopy: Absorption spectrum of zeaxanthin in various solvents (a) Hexane, (b) CHCl 3 (c) MeOH,
(d) EtAC
solvents like MeOH, DMF, DMSO (Fig.4); but in the case
of non-polar solvents, intensity is enhanced. The single
state fluorescent emission study done by Massimo crimi et
al, (2001) showed similar excitation pattern asserting the
self quenching effect of zeaxanthin present in flower
extract of T. stans .
100
90
1743.58
2675.17
549.11
1263.30
1822.27
1375.49
633.84
991.76
80
722.36
1641.24
3077.42
70
908.73
60
1463.84
%T
3452.49
50
40
30
20
10
2852.26
2923.04
0
4000
3500
3000
2500
2000
1500
1000
500
Wavenumbers (cm-1)
Fig. 4. Infrared spectrum of (a) zeaxanthin
Emission spectroscopy
The extract exhibited two well define steady state
fluorescence emission bands when exited at 420 nm
corresponding to the singlet - singlet Q0-0 and Q0-1
transitions at 650 and 715 nm respectively. The normalized
fluorescence emission of fraction in various solvent is
given in fig. 5. On comparing the fluorescence emission
among the polar and non-polar solvents, there is no shift in
the emission spectrum but the emission intensity
considerably decreases due to the self quenching in polar
11
Fig. 5. Emission spectrum of zeaxanthin in various solvent
(a) Hexane, (b) DCM (c) CHCl3, (d) MeOH, (e) DMF, (f)
DMS
International Journal of Natural Products Research 2014; 4(1): 9-11
Table 1. Fluorescence decay profile
Sample
Excitation
zeaxanthin
420
emission
650
2
X
1(A1%)
2(A1%)
3(A1%)
1.012
2.65 (32.35)
7.94 (47.62)
4.37 (20.03)
Life time studies
In order to identify the existence of different molecular
species and to obtain more quantitative information in the
exited states, fluorescence measurement was carried out for
fraction through the single photon counting technique. The
fluorescence lifetime decay profile for the fraction shown
in the fig. 6 and the fitting data and amplitudes of
fluorescent species decaying profile is found to fit with
three exponential exhibited by decaying major isomer of
zeaxanthin (table-1). The fluorescent decay profile
obtained in the present study for Zeaxanthin strongly
support the self quenching effect of the zeaxanthin reported
l by Massimo crimi et al, (2001) and Gilmore et al,(1995).
The fluorescent decay profile obtained in the present for
Zeaxanthin strongly support the self quenching effect of the
zeaxanthin reported by Massimo crimi et al, (2001) and
Gilmore et al,(1995).
Fig.6. Fluorescence decay profile of zeaxanthin in Dichloro
methane (DCM). The solid line represents best fit to I (t) =
A1exp –t/t 1 + A2exp –t/t 2 + A3exp –t/t3 Excitation (λ) = 420
nm and emission (λ) = 650 nm.
Fig. 7. a, b 1H NMR spectrum of zeaxanthin in CDCl3
12
NMR studies
1
H NMR spectrum fig.7.b shows the fraction contains
linear conjugated 14 - olefinic protons clearly demonstrates
with six different signals within the range of 5 ppm to 7.4
ppm. Doublet of doublet obtained at δ 5.01 ppm for 4
protons (8, 10, 12, and 14). Multiplet obtained for total 5
protons at δ 5.4 ppm (11, 15, 15’, 11’, 7’) and another
multiple is obtained at δ 5.86 ppm for 2 protons (11, 11’).
The appearance of three doublets at 6.61, 7.08, 7.26 ppm
respectively, for each different single proton namely for (3,
3’, 4’). In figure, 7a two broad singlets obtained at 3.66,
3.75(allylic) ppm due to –OH groups of the zeaxanthin.
Two different multiples shows for corresponding
cyclohexane rings (2.77, 2.33 ppm (a, b). The singlet
corresponds to olefin attached methyl protons at 1.57 ppm
for 18 protons. The remaining four aliphatic methyl groups
demonstrates singlet at 1.25 ppm for 120 protons. From the
1
H NMR spectral evidence shows present separated
compound is zeaxanthin.
Fig.8. 13C NMR spectrum of zeaxanthin in CDCl3
Further, the 13C-NMR spectrum fig.8 displayed at 13
different signals for extracted dye compound. The
downfield region demonstrates 7 signals namely 139.43,
130.37, 130.20, 128.20, 128.07, 122.73, 114.22 ppm for
(6), (9, 9’ 13, 13’), (1, 1’), (11, 11), (8,12), (12, 12’), (7, 7’)
carbons. The –OH attached carbons at 114.22 ppm, (3, 3’)
carbons at 51.58 ppm, (5, 2) carbon at 39.53 ppm, non olefinic carbon attached methyl groups at 29.85
ppm,conjugated carbon attached methyl groups at 17.7
ppm confirm the presence of zeaxanthin. The 1H NMR
spectral evidence and 13C-NMR spectrum data coincides
with the data provided by Duangnapa Sompong and
Pichaya Trakanrungroj, (2010) and reports from flowers of
Radermachera ignea (Kurz) by Eisenrich(2002).
Tecoma stans is found to be not only potential but also
bounteous source of natural yellow dye. Since it is a
International Journal of Natural Products Research 2014; 4(1): 9-11
invasive species, its usage as the raw material for dbio-dye
preparation is quite worthwhile. The study also revealed
that pre-mordanting the cotton cloth before dyeing yields
better color fastness where stannous chloride is used as
mordant. With regard to the spectral characteristics and the
organics with striking fluorescent nature has wide
application in the field of molecular biology. The structural
elucidation confounded that the extracted natural dye
compound is zeaxanthin with self quenching property.
Thus, the present investigation adds not only a new source
bio dye but also new source of health care pigment with
eco-friendliness and cost effectiveness.
Acknowledgement
Authors are thankful to Central Instrumentation Facility
and Department of chemistry, Pondicherry University for
their help in sample analysis. One of the authors ( A )
expresses his sincere thanks to Mr. R. Rajesh Ph.D.,
research scholar for his help in spectral analysis and
interpretation.
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Source of support: Nil; Conflict of interest: None declared
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International Journal of Natural Products Research 2014; 4(1): 9-11