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Extract Pigment
Extract Pigment
ORIGINAL ARTICLE
Department of Biotechnology, School of Bioengineering, SRM University, Kattankulathur 603203, Tamilnadu, India
KEYWORDS Abstract Recently, natural dyes and pigments gain more importance in food and textile industries
Natural pigment; because of their non toxic and eco friendly characteristics. Bougainvillea glabra oral bracts are rich
Bougainvillea glabra; in betalain pigments which can be used as a dye in sensitized solar cells, medicinal and food appli-
Floral bracts; cations. The aim of this study was to optimize the natural pigment extraction from the oral bracts
Central composite design; by response surface methodology. Central composite design (CCD) of response surface methodol-
Response surface methodol- ogy (RSM) was applied to evaluate the optimal conditions of three process variables namely mass
ogy; of oral bracts (g), extraction time (h) and temperature (C) studied at ve levels. Mass of bracts
Fourier Transform Infrared
and extraction time were found statistically signicant in the process and correlation coefcient
Spectroscopy
(R2) value of 0.96 showed that model was well tted with the experimental values. The optimum
process conditions were found to be mass of oral bracts: 3 g, contact time: 6 h and extraction tem-
perature: 22.5 C with maximum absorbance of 9.18. Response surface methodology was per-
formed well to identify the optimal levels of extraction process variables and the validation of
predicted model was tted 99.76% with the experimental results conducted at the optimum condi-
tions. Fourier Transform Infrared Spectroscopy was also conrmed the presence of betalain pig-
ment by identifying the major functional groups.
2013 Production and hosting by Elsevier B.V. on behalf of King Saud University.
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http://dx.doi.org/10.1016/j.arabjc.2013.07.047
Please cite this article in press as: Kumar, S.N.A. et al., Extraction optimization and characterization of water soluble red purple pigment from
oral bracts of Bougainvillea glabra. Arabian Journal of Chemistry (2013), http://dx.doi.org/10.1016/j.arabjc.2013.07.047
2 S.N.A. Kumar et al.
Color of the commercial products plays a vital role to at- 2.1. Collection of oral bracts
tract the consumers and also represent the quality of the
products (Azeredo, 2009; Downham and Collins, 2000). Syn- Fresh oral bracts were collected from B. glabra plant grown
thetic dyes and pigments are widely used as coloring agents in SRM University campus. Floral bracts were cut approxi-
in industries for the production of commercial products. mately into 10 mm in size and used for extraction process.
These coloring agents are a major pollutant that affects
the soil and water sources (Eichlerova et al., 2007). Waste 2.2. Pigment extraction
water generated from textile industries containing synthetic
dyes was found toxic to the aquatic eco-system (Michaels Methanol and water in 0.5:1.5 ratio were used as solvent sys-
and Lewis, 1985; Vaidya and Datye, 1982). Difculty in tem for extraction process. 50 mL of the solvent was added
treatment of textile waste water was the major issue in pres- to 250 mL Erlenmeyer ak containing known amount of the
ent days (Eichlerova et al., 2007). To overcome this prob- bracts according to the design (Table.2). Flask was tightly cov-
lem, researchers were involved in the development of ered by the polyethylene cover to avoid evaporation of the sol-
natural dyes and pigments. Various studies were reported vent. Flask was kept in a temperature controlled orbital shaker
for natural pigments derived from plant (Strack et al., for appropriate time to completely extract the pigment.
2003; Heuer et al., 1994) and microbial sources (Chiu and
Poon, 1993; Cho et al., 2002; Mukherjee and Singh, 2.3. Analytical method
2011).Several plants in nature can yield pigments which
can be used for many useful purposes (Feugang et al.,
2006; Wu et al., 2006; Vaillant et al., 2005). Bougainvillea After the extraction process, 10 mL of the solvent (contain sol-
is a genus of bright owering plants belonging to the Nycta- uble pigment) was taken in a falcon tube and centrifuged at
ginaceae family. Bougainvillea is a popular ornamental plant 5000 rpm for 10 min. The supernatant was suitably diluted
in most areas with warm climatic conditions. Bougainvillea and absorbance was measured at 538 nm by using UVVis
glabra also called as paper ower having shiny green and spectrophotometer.
magenta or purple colored bracts. Betalain pigments are
mainly responsible for color of the bract, particularly beta- 2.4. Statistical optimization
cyanins (Moreno et al., 2008; Piattelli, 1981). Betacyanins
are water soluble red- violet color pigment containing nitro- Central composite design was used to evaluate the optimum
gen in its structure (Table.1) (Moreno et al., 2008). The ma- conditions for extraction process. Statistical analysis was done
jor advantages of betalain pigments are they are independent by using Design Expert software 8.0.5.2. The three parameters
on pH and are more stable than anthocyanin pigments (Ta- selected were mass of petals (g), extraction time (h) and tem-
naka et al., 2008). Betalain pigments are having high poten- perature (C) and were studied at ve levels
tial as dye sensitizer and utilized for the photo- (+2,+1,0,1,2). Nineteen experiments were carried out
electrochemical studies (Hernandez-Martinez et al., 2011; according to the design table given in Table.2 and response
Zhang et al., 2008). pigment optical density (OD) was tabulated. A second order
Statistical optimization methods are successfully used to polynomial model given in (Eq. (1)) was used to t the ob-
identify the optimal level of various parameters involved in served data in CCD. All the experiments were performed in
the process. Optimization by changing one factor at a time triplicate to reduce the experimental errors.
(OFAT) was a common and well studied method but it has X X X
Y a0 ai Xi aii X2i aij Xi Xj 1
many disadvantages like time consuming, expensive etc. and
it does not provide details about the interaction effect of the where ai, aii, and aij are linear, quadratic and interaction effect
variables involved in the process (Haaland, 1989). Response coefcients respectively.
surface methodology (RSM) is a collection of statistical tool
used to analyze and determine the optimal conditions within 3. Results and discussion
the design space of the experimental study (Myers and Mont-
gomery, 1995). The main advantage of using RSM is to under- 3.1. Optimization by response surface methodology
stand the interaction among the process variables with less
number of experimental runs and it is utilized well for various
optimization studies (Sharmila et al., 2013; Ramandi et al., Central composite design (CCD) of RSM was used to design
2012; Baboukani et al., 2012). the experiment and nineteen experiments were carried out
The main objective of the study was to determine the according to the combination of the variables given in the de-
optimal conditions of water soluble pigment extraction pro- sign table and the observed results are given in (Table.2).
cess from B. glabra oral bracts using central composite de-
sign (CCD) of RSM. Mass of oral bract, contact time and 3.1.1. Regression analysis and model tting
temperature were chosen for the optimization study. Struc- The response, pigment OD was varied from 1.041 to 9.514 in
tural analysis of extracted pigment was also examined by the experimental runs. The observed experimental results in
FTIR. each run were subjected to multiple regression analysis to
Please cite this article in press as: Kumar, S.N.A. et al., Extraction optimization and characterization of water soluble red purple pigment from
oral bracts of Bougainvillea glabra. Arabian Journal of Chemistry (2013), http://dx.doi.org/10.1016/j.arabjc.2013.07.047
Extraction optimization and characterization of water soluble red purple pigment from oral bracts 3
Table 2 Central composite design matrix represented in actual and coded values (in brackets) with observed and predicted response.
Run Variables Pigment OD
(A) mass of bracts (g) (B) time (h) (C) temperature (C) Experimental Predicted
1 1.0 (1) 2.0 (1) 20.0 (1) 1.423 1.227
2 3.0 (+1) 2.0 (1) 20.0 (1) 2.571 2.412
3 1.0 (1) 6.0 (+1) 20.0 (1) 3.387 2.532
4 3.0 (+1) 6.0 (+1) 20.0 (1) 9.514 9.132
5 1.0 (1) 2.0 (1) 40.0 (+1) 2.351 2.283
6 3.0 (+1) 2.0 (1) 40.0 (+1) 1.793 2.198
7 1.0 (1) 6.0 (+1) 40.0 (+1) 1.846 1.555
8 3.0 (+1) 6.0 (+1) 40.0 (+1) 7.140 6.886
9 0.3 (2) 4.0 (0) 30.0 (0) 1.148 1.726
10 3.7 (+2) 4.0 (0) 30.0 (0) 7.232 7.264
11 2.0 (0) 0.6 (2) 30.0 (0) 1.046 0.774
12 2.0 (0) 7.4 (+2) 30.0 (0) 5.036 5.868
13 2.0 (0) 4.0 (0) 13.2 (2) 2.764 3.496
14 2.0 (0) 4.0 (0) 46.8 (+2) 2.582 2.496
15 2.0 (0) 4.0 (0) 30.0 (0) 5.382 5.127
16 2.0 (0) 4.0 (0) 30.0 (0) 4.842 5.127
17 2.0 (0) 4.0 (0) 30.0 (0) 4.656 5.127
18 2.0 (0) 4.0 (0) 30.0 (0) 4.938 5.127
19 2.0 (0) 4.0 (0) 30.0 (0) 5.924 5.127
Please cite this article in press as: Kumar, S.N.A. et al., Extraction optimization and characterization of water soluble red purple pigment from
oral bracts of Bougainvillea glabra. Arabian Journal of Chemistry (2013), http://dx.doi.org/10.1016/j.arabjc.2013.07.047
4 S.N.A. Kumar et al.
Figure 1 3D surface plots showing the mutual effect between Figure 3 3D surface plots showing the combined effect between
pair of variables, mass of oral bracts (A) and time (B) by keeping pair of variables, time (B) and temperature (C) by keeping the
the third variable constant at middle level. third variable constant at middle level.
Please cite this article in press as: Kumar, S.N.A. et al., Extraction optimization and characterization of water soluble red purple pigment from
oral bracts of Bougainvillea glabra. Arabian Journal of Chemistry (2013), http://dx.doi.org/10.1016/j.arabjc.2013.07.047
Extraction optimization and characterization of water soluble red purple pigment from oral bracts 5
Figure 4 FTIR analysis spectrum for the redpurple pigment extracted from oral bracts of B. glabra.
predicted optical density of 9.18. This result explained that the 22.5 C with maximum optical density of 9.18. Validation of
predicted model had a good agreement with the experimental the predicted model was tted 99.76% with the experimental
results. results conducted at the optimum conditions. FTIR analysis
of pigment showed the presence of the nitrogen (NH) amine
3.2. FTIR analysis group and conrmed that the extracted pigment belongs to
betacyanin of betalains.
The Fourier Transform Infrared (FTIR) spectral analysis was
done to indentify the major functional groups present in the
extracted pigment (DeSouza et al., 2003). FTIR spectrum Acknowledgments
represented in Fig. 4 shows distinct peaks at 3394, 2953,
2842, 2527, 2148, 1651, 1451, 1412, 1115, 1018 and Authors wish to thank the Management, Director (E&T) and
718 cm1 respectively. The broad and strong band at Department of Biotechnology for providing necessary facilities
3394 cm1 suggests (O-H) bond in stretching vibration mode to carry out this study and also acknowledges The Sophisti-
and band at 2953 and 2842 cm1 indicates (CH) symmetry cated Analytical Instrument Facility (SAIF) at IIT, Madras
in stretching mode. The wave numbers 2527 and 2148 cm1 for FTIR analysis.
showed the presence of (OH) and (CC) in stretching
vibration mode respectively. The wave number 1651 cm1
conrmed the presence of the carbonyl group (CO) in References
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oral bracts of Bougainvillea glabra. Arabian Journal of Chemistry (2013), http://dx.doi.org/10.1016/j.arabjc.2013.07.047
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Please cite this article in press as: Kumar, S.N.A. et al., Extraction optimization and characterization of water soluble red purple pigment from
oral bracts of Bougainvillea glabra. Arabian Journal of Chemistry (2013), http://dx.doi.org/10.1016/j.arabjc.2013.07.047