Arabian Journal of Chemistry (2017) 10, S2145–S2150

King Saud University

Arabian Journal of Chemistry

www.ksu.edu.sa
www.sciencedirect.com

ORIGINAL ARTICLE

Extraction optimization and characterization

of water soluble red purple pigment from floral
bracts of Bougainvillea glabra
Shiv Narayan Amit Kumar, Sunil Kumar Ritesh, Govindasamy Sharmila,
Chandrasekaran Muthukumaran *

Department of Biotechnology, School of Bioengineering, SRM University, Kattankulathur 603203, Tamilnadu, India

Received 29 October 2012; accepted 21 July 2013

Available online 27 July 2013

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 floral 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 floral 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 floral bracts (g), extraction time (h) and temperature (C) studied at five levels. Mass of bracts
Fourier Transform Infrared
and extraction time were found statistically significant in the process and correlation coefficient
Spectroscopy
(R2) value of 0.96 showed that model was well fitted with the experimental values. The optimum
process conditions were found to be mass of floral 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 fitted 99.76% with the experimental results conducted at the optimum condi-
tions. Fourier Transform Infrared Spectroscopy was also confirmed the presence of betalain pig-
ment by identifying the major functional groups.
ª 2013 Production and hosting by Elsevier B.V. on behalf of King Saud University. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Abbreviations: ANOVA, analysis of variance, CCD, central composite

design, FTIR, Fourier Transform Infrared Spectroscopy, OD, optical
density, OFAT, one factor at a time, RSM, response surface
methodology
* Corresponding author. Tel.: +91 44 27452270; fax: +91 44
27453903.
E-mail address: biopearl1981@gmail.com (C. Muthukumaran).
Peer review under responsibility of King Saud University.

Production and hosting by Elsevier

http://dx.doi.org/10.1016/j.arabjc.2013.07.047
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This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
S2146
1. Introduction
Color of the commercial products plays a vital role to at-
tract the consumers and also represent the quality of the
products (Azeredo, 2009; Downham and Collins, 2000). Syn-
thetic dyes and pigments are widely used as coloring agents
in industries for the production of commercial products.
These coloring agents are a major pollutant that affects
the soil and water sources (Eichlerova et al., 2007). Waste
water generated from textile industries containing synthetic
dyes was found toxic to the aquatic eco-system (Michaels
and Lewis, 1985; Vaidya and Datye, 1982). Difficulty in
treatment of textile waste water was the major issue in pres-
ent days (Eichlerova et al., 2007). To overcome this prob-
lem, researchers were involved in the development of
natural dyes and pigments. Various studies were reported
for natural pigments derived from plant (Strack et al.,
2003; Heuer et al., 1994) and microbial sources (Chiu and
Poon, 1993; Cho et al., 2002; Mukherjee and Singh,
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
is a genus of bright flowering plants belonging to the Nycta-
ginaceae family. Bougainvillea is a popular ornamental plant
in most areas with warm climatic conditions. Bougainvillea
glabra also called as paper flower having shiny green and
magenta or purple colored bracts. Betalain pigments are
mainly responsible for color of the bract, particularly beta-
cyanins (Moreno et al., 2008; Piattelli, 1981). Betacyanins
are water soluble red- violet color pigment containing nitro-
gen in its structure (Table.1) (Moreno et al., 2008). The ma-
jor advantages of betalain pigments are they are independent
on pH and are more stable than anthocyanin pigments (Ta-
naka et al., 2008). Betalain pigments are having high poten-
tial as dye sensitizer and utilized for the photo-
electrochemical studies (Hernandez-Martinez et al., 2011;
Zhang et al., 2008).
Statistical optimization methods are successfully used to
identify the optimal level of various parameters involved in
the process. Optimization by changing one factor at a time
(OFAT) was a common and well studied method but it has
many disadvantages like time consuming, expensive etc. and
it does not provide details about the interaction effect of the
variables involved in the process (Haaland, 1989). Response
surface methodology (RSM) is a collection of statistical tool
used to analyze and determine the optimal conditions within
the design space of the experimental study (Myers and Mont-
gomery, 1995). The main advantage of using RSM is to under-
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.,
2017; Baboukani et al., 2012).
The main objective of the study was to determine the
optimal conditions of water soluble pigment extraction pro-
cess from B. glabra floral bracts using central composite de-
sign (CCD) of RSM. Mass of floral bract, contact time and
temperature were chosen for the optimization study. Struc-
tural analysis of extracted pigment was also examined by
FTIR.
S.N.A. Kumar et al.
2. Materials and methods
2.1. Collection of floral bracts
Fresh floral bracts were collected from B. glabra plant grown
in SRM University campus. Floral bracts were cut approxi-
mately into 10 mm in size and used for extraction process.
2.2. Pigment extraction
Methanol and water in 0.5:1.5 ratio were used as solvent sys-
tem for extraction process. 50 mL of the solvent was added
to 250 mL Erlenmeyer flak containing known amount of the
bracts according to the design (Table.2). Flask was tightly cov-
ered by the polyethylene cover to avoid evaporation of the sol-
vent. Flask was kept in a temperature controlled orbital shaker
for appropriate time to completely extract the pigment.
2.3. Analytical method
After the extraction process, 10 mL of the solvent (contain sol-
uble pigment) was taken in a falcon tube and centrifuged at
5000 rpm for 10 min. The supernatant was suitably diluted
and absorbance was measured at 538 nm by using UV–Vis
spectrophotometer.
2.4. Statistical optimization
Central composite design was used to evaluate the optimum
conditions for extraction process. Statistical analysis was done
by using Design Expert software 8.0.5.2. The three parameters
selected were mass of petals (g), extraction time (h) and tem-
perature (C) and were studied at five levels
(+2,+1,0,1,2). Nineteen experiments were carried out
according to the design table given in Table.2 and response
pigment optical density (OD) was tabulated. A second order
polynomial model given in (Eq. (1)) was used to fit the ob-
served data in CCD. All the experiments were performed in
triplicate to reduce the experimental errors.
X X X
Y ¼ a0 þ ai Xi þ aii X2i þ aij Xi Xj ð1Þ
where ai, aii, and aij are linear, quadratic and interaction effect
coefficients respectively.
3. Results and discussion
3.1. Optimization by response surface methodology
Central composite design (CCD) of RSM was used to design
the experiment and nineteen experiments were carried out
according to the combination of the variables given in the de-
sign table and the observed results are given in (Table.2).
3.1.1. Regression analysis and model fitting
The response, pigment OD was varied from 1.041 to 9.514 in
the experimental runs. The observed experimental results in
each run were subjected to multiple regression analysis to
Extraction optimization and characterization of water soluble red purple pigment from floral bracts S2147

square effect of mass of bracts (A2) were found to be statisti-

Table 1 Structure of betalain pigments.
cally insignificant in extract process (p > 0.05). The model
Pigment name structure for pigment extraction process was modified and represented
Betanidin -
in (Eq. (3)) by neglecting the insignificant model terms.
HO COO
N
+ YPigment OD ¼ 5:13 þ 1:63A þ 1:50B þ 1:35AB  0:63B2
HO  0:76C2 ð3Þ
The linear effect of floral bract mass and time (p < 0.05), qua-
N dratic effect of extraction time (B2), temperature (C2)
HOOC H (p < 0.05), and combined effect of bract mass with time
(AB) (p < 0.05) showed significance on pigment extraction.
High F-test value of floral bract mass (82.04) and time
Betanin -
Beta-D-glucose -O COO (69.41) of linear effect explained that these variables contribute
+ more on extraction than other effects. Determination coeffi-
N
HO cient (R2) value near to unity denotes the quality of the good
fit (Weisberg, 1985). Determination coefficient (R2) value of
0.96 revealed that the regression model was statistically signif-
N icant and 0.04% of variation was not explained by the model.
HOOC H
3.1.2. Surface plots
The 3D surface plots represented in Figs. 1–3 were used to
study the interaction among the variables in the process (Bas
and Boyaci, 2007). Combined effect of floral bract mass and
time represented in Fig. 1 explained that the pigment absor-
calculate the regression coefficients of the model (Eq. (1)). Cal-
bance was increased as the time increases and remains constant
culated regression coefficients were substituted in (Eq. (1)) to
with mass of floral bracts. Similar trend was reported by Sinha
obtain a model (Eq. (2)) for the extraction of water soluble pig-
et al. (2012) on natural dye extraction from petals of Flame of
ment from bracts.
forest flower. Maximum absorbance of pigment (8.74) was ob-
YPigment OD ¼ 5:13 þ 1:63A þ 1:50B  0:30C þ 1:35AB served at a high level of time (6 h) and bract mass (2.99 g).Fig. 2
 0:38AC  0:51BC  0:22A2  0:63B2 shows the mutual effect of mass of floral bract and tempera-
ture. Pigment OD was gradually increased from low to high le-
 0:76C2 ð2Þ vel of floral bract mass whereas it remain constant with the
Results of the regression analysis tabulated in Table.3, ex- temperature. The optimal OD value of 6.658 was expected at
plained that linear effect of temperature (C), interaction effect 25.5 C and 3 h of contact time. Interaction between contact
of temperature with mass of bracts (AC) and time (BC), and time and temperature was explained by Fig. 3, where the high

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
S2148 S.N.A. Kumar et al.

Table 3 ANOVA table for pigment OD.

Source Pigment OD
Coefficient Sum of squares DF Mean square F value p-Value prob > F
Model 5.13 97.13 9 10.79 24.44 <0.0001a
(A) mass of bracts 1.63 36.23 1 36.23 82.04 <0.0001 a
(B) time 1.50 30.66 1 30.66 69.41 <0.0001 a
(C) temperature 0.30 1.21 1 1.21 2.75 0.1318
AB 1.35 14.66 1 14.66 33.20 0.0003 a
AC 0.38 0.81 1 0.81 1.82 0.2098
BC 0.51 2.07 1 2.07 4.68 0.0588
A2 0.22 0.65 1 0.65 1.48 0.2551
B2 0.63 5.33 1 5.33 12.07 0.0070 a
C2 0.76 7.78 1 7.78 17.62 0.0023 a
Residual 3.97 9 0.44
Lack of fit 2.94 5 0.59 2.27 0.2239
Pure error 1.04 4 0.26
Cor total 101.10 18
R2 0.960
a
Significant model parameters (p < 0.05).

OD value (6.214) was observed when time and temperature

were maintained at 6 h and 24.4 C respectively.
The point prediction tool of the Design Expert software
was used to identify the optimal value of the chosen process
variables. Optimal values of floral bract mass (A), contact time
(B) and extraction temperature (C) were found to be 3 g, 6 h
and 22.5 C respectively with maximum predicted response
of 9.18.

3.1.3. Model validation

The validation experiment for the predicted optimum condi-
tions was performed in triplicate to check the accuracy of
the model. Three grams of bract was taken in the 250 mL flask
containing 50 mL of the selected solvent (methanol:water:
0.5:1.5). The flask was kept at 22.5 C for 6 h and the optical
density of the extracted pigment was taken at 538 nm. The ob-
Figure 2 3D surface plots representing the interaction effect
tained optical density was found to be 9.16 which proved the
between pair of variables, mass of floral bract (A) and temperature
model to be 99.76% accurate when compared with the
(C) by keeping the third variable constant at middle level.

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 floral 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.
Extraction optimization and characterization of water soluble red purple pigment from floral bracts
Figure 4 FTIR analysis spectrum for the red–purple pigment extracted from floral bracts of B. glabra.
predicted optical density of 9.18. This result explained that the
predicted model had a good agreement with the experimental
results.
3.2. FTIR analysis
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
represented in Fig. 4 shows distinct peaks at 3394, 2953,
2842, 2527, 2148, 1651, 1451, 1412, 1115, 1018 and
718 cm1 respectively. The broad and strong band at
3394 cm1 suggests (O-H) bond in stretching vibration mode
and band at 2953 and 2842 cm1 indicates (C–H) symmetry
in stretching mode. The wave numbers 2527 and 2148 cm1
showed the presence of (O–H) and (C„C) in stretching
vibration mode respectively. The wave number 1651 cm1
confirmed the presence of the carbonyl group (C‚O) in
stretching mode associated with amide bond. The weak peak
at 1451 and 1412 cm1 suggested the presence of bending
aromatic (C‚C) bond. The peaks at 1115 and 1018 cm1
represented the (C–O) bond and the presence of the amine
group (N–H) was confirmed by the wave number 718 cm1
in the spectrum. The presence of amine (N–H) and carbonyl
(C‚O) and other functional groups confirmed that the ex-
tracted red–purple pigment from B. glabra belongs to the
betacyanin group of betalains.
4. Conclusion
This study explained the utilization of B. glabra floral bracts
for successful extraction of natural pigment. Mass of the floral
bracts and extraction time play an important role to increase
the efficiency of pigment extraction at higher levels. Experi-
mental results revealed that the extraction of pigment was
highly favored at low temperatures than at elevated levels.
The optimum process conditions were found to be mass of flo-
ral bracts: 3 g, contact time: 6 h and extraction temperature:
S2149
22.5 C with maximum optical density of 9.18. Validation of
the predicted model was fitted 99.76% with the experimental
results conducted at the optimum conditions. FTIR analysis
of pigment showed the presence of the nitrogen (N–H) amine
group and confirmed that the extracted pigment belongs to
betacyanin of betalains.
Acknowledgments
Authors wish to thank the Management, Director (E&T) and
Department of Biotechnology for providing necessary facilities
to carry out this study and also acknowledges The Sophisti-
cated Analytical Instrument Facility (SAIF) at IIT, Madras
for FTIR analysis.
References
Azeredo, H.M.C., 2009. Betalains: properties, sources, applications,
and stability–a review. Int. J. Food Sci. Technol. 44, 2365–2376.
Baboukani, B.S., Vossoughi, M., Alemzadeh, I., 2012. Optimisation of
dilute-acid pretreatment conditions for enhancement sugar recov-
ery and enzymatic hydrolysis of wheat straw. Biosys. Eng. 111,
166–174.
Bas, D., Boyaci, I.H., 2007. Modeling and optimization I: usability of
response surface methodology. J. Food Eng. 78, 836–845.
Chiu, S.W., Poon, Y.K., 1993. Submerged production of Monascus
pigments. Mycologia 85, 214–218.
Cho, Y.J., Park, J.P., Hwang, H.J., Kim, S.W., Choi, J.W., Yun, J.W.,
2002. Production of red pigment by submerged culture of Paeci-
lomyces sinclairii. Lett. Appl. Microbiol. 35, 195–202.
DeSouza, R.F.V., Sussuchi, E.M., De Giovani, W.F., 2003. Synthesis,
electrochemical, spectral, and antioxidant properties of complexes
of flavonoids with metal ions. Synth. React. Inorg. Met. Org.
Chem. 33, 1125–1144.
Downham, A., Collins, P., 2000. Colouring our foods in the last and
next millennium. Int. J. Food Sci. Technol. 35, 5–22.
Eichlerova, I., Homolka, L., Nerud, F., 2007. Decolorization of high
concentrations of synthetic dyes by the white rot fungus Bjerkan-
dera adusta strain CCBAS 232. Dyes Pigm. 75, 38–44.
S2150
Feugang, J.M., Konarski, P., Zou, D., Stintzing, F.C., Zou, C., 2006.
Nutritional and medicinal use of cactus pear (Opuntia spp.)
cladodes and fruits. Front. Biosci. 11, 2574–2589.
Haaland, P.D., 1989. Experimental Design in Biotechnology. Dekker,
New York.
Hernandez-Martinez, A.R., Estevez, M., Vargas, S., Quintanilla, F.,
Rodriguez, R., 2011. New dye-sensitized solar cells obtained from
extracted bracts of Bougainvillea glabra and Spectabilis betalain
pigments by different purification processes. Int. J. Mol. Sci. 12,
5565–5576.
Heuer, S., Richter, S., Metzger, J.W., Wray, V., Nimtz, M., Strack, D.,
1994. Betacyanins from bracts of Bougainvillea glabra. Phytochem-
istry 37, 761–767.
Michaels, G.B., Lewis, D.L., 1985. Sorption and toxicity of azo and
triphenylmethane dyes to aquatic microbial populations. Environ.
Toxicol. Chem. 4, 45–50.
Moreno, D.A., Garcı́a-Viguera, C., Gil, J.I., Gil-Izquierdo, A., 2008.
Betalains in the era of global agri-food science, technology and
nutritional health. Phytochem. Rev. 7, 261–280.
Mukherjee, G., Singh, S.K., 2011. Purification and characterization of
a new red pigment from Monascus purpureus in submerged
fermentation. Process Biochem. 46, 188–192.
Myers, R.H., Montgomery, D.C., 1995. Response Surface Methodol-
ogy, Process and Product Optimization Using Design Experiments.
John Wiley & Sons, New York.
Piattelli, M., 1981. The betalains: structure, biosynthesis, and chemical
taxonomy. In: Conn, E.E. (Ed.), The Biochemistry of Plants.
Academic Press, New York, pp. 557–575.
S.N.A. Kumar et al.
Ramandi, N.F., Ghassempour, A., Najafi, N.M., Ghasemi, E., 2017.
Optimization of ultrasonic assisted extraction of fatty acids from
Borago officinalis L. flower by central composite design. Arabian J.
Chem. 10, S23–S27.
Sharmila, G., Nidhi, B., Muthukumaran, C., 2013. Sequential statistical
optimization of red pigment production by Monascus purpureus
(MTCC 369) using potato powder. Ind. Crop. Prod. 44, 158–164.
Sinha, K., Saha, P.D., Datta, S., 2012. Extraction of natural dye from
petals of Flame of forest Butea monosperma flower: process
optimization using response surface methodology RSM. Dyes
Pigm. 94, 212–216.
Strack, D., Vogt, T., Schliemann, W., 2003. Recent advances in
betalain research. Phytochemistry 62, 247–269.
Tanaka, Y., Sasaki, N., Ohmiya, A., 2008. Biosynthesis of plant
pigments: anthocyanins, betalains and carotenoids. Plant J. 54,
733–749.
Vaidya, A.A., Datye, K.V., 1982. Environmental pollution during
chemical processing of synthetic fibres. Colourage 14, 3–10.
Vaillant, F., Perez, A., Davila, I., Dornier, M., Reynes, M., 2005.
Colourant and antioxidant properties of red-purple pitahaya
(Hylocereus sp.). Fruits 60, 1–10.
Weisberg, S., 1985. Applied Linear Regression. Wiley, New York.
Wu, L., Hsu, H.W., Chen, Y.C., Chiu, C.C., Lin, Y.I., Ho, J.A., 2006.
Antioxidant and antiproliferative activities of red pitaya. Food
Chem. 95, 319–327.
Zhang, D., Lanier, S.M., Downing, J.A., Avent, J.L., Lum, J.,
McHale, J.L., 2008. Betalain pigments for dye-sensitized solar cells.
J. Photochem. Photobiol. A: Chem. 195, 72–80.