Extraction and Quantification of Anthocyanin from Banana Bracts Using Different pH and Solvent Concentration

Extraction and Quantification of Anthocyanin from Banana Bracts Using Different pH and Solvent Concentration
Extraction and Quantification of Anthocyanin from Banana
Bracts Using Different pH and Solvent Concentration
T.A. Ove1, *M.M. Kamal2, S.M.N.I. Nasim3, M.M.I Momin4, S.C. Mondal5
1,2,3,4,5Department of Food Processing and Preservation, Faculty of Engineering, Hajee Mohammad Danesh Science and
Technology University, Dinajpur-5200, Bangladesh
Banana (Musa balbisiana) bracts were investigated as a potential source of natural colorant. In
this study, the total anthocyanin from selected banana bracts was extracted with ethanol
solutions and characterized by UV-visible spectrophotometry and their content was found 224.41
± 1.91 mg/kg, which was highest at 40% solvent concentration with pH 4. Moreover, the color
characteristics were varied with the variation of solvent concentration and pH. Similarly, the
values of chroma and hue angle were also investigated and the value of chroma was higher at pH
4 in all different concentrations. The results revealed that the color of anthocyanin was decreased
due to increase of pH. The value of hue angle was in the range of (73.69±0.33) to (-71.14±1.39),
which indicated the color from yellow to magenta, and this is the natural color of anthocyanin.
Therefore, banana bracts can be used as a potential source of extracting natural colorant instead
of synthetic dyes in different food industries.
Keywords: Banana bracts, Anthocyanin, Extraction, Quantification, Solvent Concentration, pH
INTRODUCTION
Nowadays the use of natural colorants replaces the use of
synthetic dyes due to safety issues. According to the
numbering system used by the Codex Alimentarius
Commission, anthocyanins (any anthocyanin-derived
colorant) are listed as a natural colorant by the European
Union (EU) legislation as product E163 (Markakis, 1982).
By-products of some industrial processing can be the best
putative commercial sources of anthocyanins such as
grape skin extracts (Jackman and Smith 1996). For that
reason, more attention has been paid to other potential
anthocyanin-rich waste by-products, i.e. banana bracts
(Pazmino-Duran et al., 2001).
Various types of extraction methods have been employed
to extract anthocyanin content, among them two main
methods that have been used frequently are reflux system
(Sharifi and Hassani, 2012) and two-phase aqueous
system (Hua et al., 2013). Different solvents had been
used during extraction such as acidified water, acidified
methanol, ethanol, acetone and acid such as hydrochloric
acid (HCL) (Chandrasekhar et al., 2012). During the
extraction of anthocyanins, aqueous acidified methanol
and ethanol have been most commonly used (Fan et al.,
2008; Bridgers et al., 2010; Chandrasekhar et al., 2012;
Truong et al., 2012; Kang et al., 2013; Puertolas et al.,
2013).
Researchers found that the degree of extraction of
anthocyanin was the highest in the case of acetone
followed by acidified methanol and acidified ethanol
(Chandrasekhar et al., 2012). However, scientists also
found that the use of acetone and methanol in the food
industry is not preferable because of their possible toxicity
(Spagna et al., 2003; Patil et al., 2009). Ethanol is the most
acceptable one for use in food industry (Patil et al., 2009;
Bridgers et al., 2010; Truong et al., 2010; Lu et al., 2011;
Chandrasekhar et al., 2012; Truong et al., 2012; Burgos et
al., 2013; Kang et al., 2013).
*Corresponding Author: M.M. Kamal, Department of
Food Processing and Preservation, Faculty of
Engineering, Hajee Mohammad Danesh Science and
Technology University, Dinajpur-5200, Bangladesh. E-
mail: murtuzakamal@gmail.com
Co-Authors Email: 1
towkirahmedove@gmail.com
3
nasimalam71@yahoo.com; 4
momin.fpe12@gmail.com
5
shakti.c.mondal@hstu.ac.bd
Research Article
Vol. 4(2), pp. 060-064, April, 2019. © www.premierpublishers.org. ISSN: 2167-0434
International Journal of Food and Nutrition Sciences

Kamal et al. 061
Hence, ethanol can be considered as a potential one for
application of food. In order to extract hydrophilic
anthocyanins, scientists suggested that pure ethanol
should not be used. According to Patil et al. (2009) little
amount of water can be added with ethanol for extraction.
Moreover, various factors influence the stability of
anthocyanin pigments such as pH value, temperature, the
presence of oxygen, enzymes, metal ions and so on
(Andersen et al., 2008). Researchers also found that
stability and color intensity was better at low pH values.
So, the protocol performed at high acidity, anthocyanin
gradually loses the color. However, this color loss is
reversible and the red hue will return to acidification (Zhao
et al., 2004; Borkowski et al., 2005).
The pH has a significant impact on the anthocyanin
molecules. In acidic media, at low pH values, anthocyanins
are being more stable than the alkaline solution with high
pH values (Rein, 2005). In acidic aqueous solution,
anthocyanins exist as four main equilibrium species: the
quinoidal base (QB), the flavylium cation (FC), the carbinol
or pseudobase (PB) and the chalcone (CH) (Wilska-
Jeszka, 2007; Bobbio and Mercadante, 2008). pH below
2, anthocyanins exist basically in flavylium cation in red or
purple. The quinoidal blue species are predominant by
increasing pH up to 4. Due to hydration of flavylium cation
colorless species carbinol or pseudobase and yellowish
chalcone exist at pH between 5 and 6 (Kahkonen and
Heinonen, 2003; Castaneda-Ovando et al., 2009).
In Bangladesh, banana bracts are usually thrown away as
a waste during the harvesting season. Recently,
anthocyanin pigments in banana bracts are found
considering a potential food colorant. As the banana bracts
are widely available and have been used as food, they
could be a potential source of anthocyanins (Pazmino-
Duran et al., 2001). Thus, the objectives of this research
were to extract the total anthocyanin from banana bracts
and to evaluate the effects of various extraction conditions
such as pH and solvent concentrations.
MATERIALS AND METHODS
Sample Collection
Banana bracts were collected from the local market in
Dinajpur and some were collected from local banana
orchard.
Apparatus for Extraction
pH meter, Visible spectrophotometer and Whatman filter
paper (no.1).
Chemicals and Reagents
Ethanol, Hydrochloric Acid (HCl), Sodium Hydroxide
(NaOH) and Distilled water.
Sample Preparation
Banana bracts were washed with tap water to remove
adherences, dirt and other surface impurities properly.
Then they were cut into small or desired pieces manually.
Solvent Preparation
The Solvents were prepared into 30%, 40% and 50%
concentration of ethanol with the addition of distilled water
in different 1000 ml beaker respectively. The pH of the
solvent solutions were maintained at 4, 5 and 6 for each
concentrations with the help of hydrochloric acid and
sodium hydroxide.
Extraction of Anthocyanin
The extraction was done by mixing 50 g of banana bracts
in pieces into 500 ml of ethanol at different concentrations
(30, 40 and 50%). The extraction process was carried out
in water bath at 50°C for 60 minutes. (Ninh et al., 2015 and
Simona et al., 2012). Each mixture was filtered through a
muslin cloth to remove coarse particles. Then vacuum
filtration with whatman filter paper (no.1) was performed to
remove other dissolved minute particles. Finally, the
filtrated extracts were used for the determination of total
anthocyanin content.
Determination of Anthocyanin Content
On the basis of extractability results, a simple, rapid
method for determining the total anthocyanin content was
established (Abdel et al., 1999). The filtrated banana bract
solution was taken for determining the absorbance at 530
nm using a spectrophotometer. The anthocyanin content
was calculated as cyanidin-3-glucoside on the basis of the
following equation:
Anthocyanin Content (mg / kg) = 6A V MW
10 .......(1)
W 1000
 

 
Where, A= Absorbance, MW= Molecular weight of
cyanidin-3-glucoside (C12H21ClO11, 449.2), V= Volume of
solvent, ɛ= Molar absorptivity (25965 cm-1 M-1), W=
Sample weight
Determination of Anthocyanin Color Intensity
The instrumental color property measurement of
anthocyanin extracts were carried out with a colorimeter
Minolta CM-2500d (Konica Minolta optics, Inc. Japan).
Color attributes were recorded as L (Lightness), a
(Redness) and b (yellowness). Chroma and Hue angle of
the specimens were evaluated using the following formula.
Chroma = 2 2
a b ...................................(2)+
Hue = tan 1 a
...................................(3)
b
−

Int. J. Food Nutr. Sci. 062
Statistical Analysis
All collected data’s were statistically analyzed by using
IBM SPSS (version 20) to obtain the mean values with
standard deviations.
RESULTS AND DISCUSSION
Anthocyanin Content
The total anthocyanin content of banana bracts ethanolic
extracts is given in table 1. In agreement with Patil et al.
(2009) pure ethanol should not be used for extraction of
anthocyanin, based on this little amount of water was used
to extract the hydrophilic anthocyanins. The anthocyanin
content was found more appreciable at 40% solvent
concentration and it was gradually decreased with
increasing the solvent concentration. However, it showed
that at 30% solvent concentration the anthocyanin
extraction was lower than that of other concentrations. The
decreasing rate of anthocyanin extraction above 40% (v/v)
ethanol concentration could be due to the non-extraction
of hydrophilic anthocyanins as the concentration of water
in the extraction media decreased with increased ethanol
content. Similarly, like solvent concentration the amount of
total anthocyanin contents was the highest at pH 4 among
three different solvent concentrations.
As it can be seen in table 1, total anthocyanin content was
significant (224.41±1.91 mg/kg) at 40% solvent
concentration with pH 4. This anthocyanin content was
closer to what was reported by Timberlake (1988) for red
cabbage (250 mg/kg), already commercially available as a
food color extract. This obtained result is higher than the
findings of Roobha et al., (2011) which was extracted from
Musa acuminata bract and slightly lower from the
optimized results of Begum and Deka (2017) for spray-
dried microencapsulated anthocyanins extracted from
culinary banana bracts i.e. (56.98 mg/100g).
Mathematically each banana plant contains about (≈) 600
g of colored bracts and per hectare produces around 1500
plants (Pazmino-Duran et al., 2001). Consequently, 1500
plants contain (600 g × 1500) 900,000 g colored bracts. In
line with our research from 900,000 g colored bracts would
be yielded (224.41 mg/kg × 900,000 g) 202 g of
anthocyanin per hectare. For that reason, it could be
commercially feasible to produce pigment from this
source.
Color Attributes of Anthocyanin Extract from Banana
Bracts
Quantitative color measurement of colorimeter Minolta
CM-2500d (Konica Minolta optics, Inc. Japan) L, a and b
are commonly used to measure the color component from
the colored solution of banana bracts. The value of L, a, b,
Chroma and Hue values of anthocyanin extracts from
colored solution of banana bracts at various condition are
shown in table 2.
Color Attributes at 30% Solvent Concentration
The characteristics of L, a, b, Chroma and Hue for 30%
solvent concentration at various pH (4, 5 and 6) were
shown in table 2. It was found that the value of L, which
indicates the lightness was increased to increasing pH and
the value of a, which indicates the redness (+) or
greenness (-) was decreased that means the redness was
turned to greenness with increasing pH. Similarly, the
value of b, which indicates yellowness (+) or blueness (-)
decreasing at increasing pH. Chroma which increased at
lower pH means that the colorfulness of anthocyanin was
brighter than elevated pH. Whether the hue angle at pH 4,
5 and 6 were (21.96±1.42), (-13.67±4.29) and
(73.69±0.33) respectively. This situation means that at pH
4 and 5 the hue angle remained from yellow to crimson
that indicates the natural color of anthocyanin but at pH 6
color turned into greenish-yellow. At elevated pH values
anthocyanins will provide color fading of colorless, yellow,
purple and blue (Wahyuningsih et al., 2016).
At 40% solvent concentration the value of L, was increased
to increasing pH and the value of a, which indicates the
redness (+) or greenness (-) was decreased which means
the redness was turned to greenness with increasing pH.
Similarly, the value of b, which indicates yellowness (+) or
blueness (-) decreasing at increasing pH.
Colorfulness of anthocyanin was brighter at lower pH than
elevated pH as the values of chroma gradually decreased
at higher pH. At pH 4, 5 and 6 the values of hue angle were
(-19.54±2.09), (-52.74±5.06) and (-67.12±8.29)
respectively. The hue angle remained in the range of fully
red color at pH 4 and at pH 5, at pH 6 the hue angle
remained from red to magenta that indicates the natural
color of anthocyanin. Lower hue angle indicates more red
color of anthocyanins. This means at higher pH values the
color of anthocyanin gradually decreased (Ibrahim et al.,
2011). In brief, lower hue angle indicated the total
anthocyanin content can be higher at lower pH.
The value of L, which indicates the lightness was
increased to increasing pH and the value a, of which
indicates the redness (+) or greenness (-) was decreased
that means the redness was turned to greenness with
increasing pH. Similarly, the value of b, which indicates
yellowness (+) or blueness (-) decreasing at increasing pH.
At lower pH the value of Chroma indicates the increased
brightness means that the colorfulness of anthocyanin was
brighter than elevated pH. The hue angle at pH 4, 5 and 6
were (-25.89±4.08), (-69.51±2.41), (-71.14±1.39)
respectively. The value at pH 4 for the hue angle remained
at a range of fully red color indicating the natural color of
anthocyanin, at pH 5 and 6 the hue angle remained to
magenta that indicates anthocyanin loses its intensity and

Kamal et al. 063
changes its color content at elevated pH. Higher pH is
associated with the development of anthocyanin
degradation (Ibrahim et al., 2011). This color change is
reversible and the red hue will return to acidification (Zhao
et al., 2004; Borkowski et al., 2005).
Table 1: Total anthocyanin content at various solvent
concentration and pH
Solvent
Concentration
pH Anthocyanin (mg/kg)
C30%
4 122.96 ± 0.31a
5 8.35 ± 0.56b
6 6.84 ±0.31c
A40%
4 224.41±1.91a
5 20.06±0.35b
6 17.84±0.35b
B50%
4 192.93±0.60a
5 19.53±0.18b
6 18.22±1.76b
All values are means ±SD of three replicates.
a-cThe test values along the same column carrying
different superscripts are significantly different (p<
0.05).
A-CThe concentration are significantly different (p<
0.05).
Table 2: Color parameters of extracted anthocyanin
from banana bracts
Solvent
Concentration
pH L a b Chroma Hue
4
28.19
±3.22b
21.04
±3.46a
8.51
±1.74a
22.7
±3.83a
21.96
±1.42b
30% 5
44.23
±5.61a
2.25
±0.12b
-0.55
±0.15c
2.32
±0.07b
-13.67
±4.29c
6
46.05
±2.51a
1.7
±0.05b
5.81
±0.14b
6.05
±0.14b
73.69
±0.33a
4
39.82
±1.24a
17.52
±2.68a
-6.16
±0.51b
18.58
±2.64a
-19.54
±2.09a
40% 5
40.56
±3.01a
2.28
±0.32b
-2.99
±0.17a
3.77
±0.13b
-52.74
±5.06b
6
45.11
±5.13a
0.84
±0.14b
-2.14
±0.81a
2.31
±0.78b
-67.12
±8.29c
4
40.93
±5.38b
11.32
±1.99a
-5.41
±0.22b
12.59
±1.76a
-25.89
±4.08a
50% 5
49.93
±0.42a
1.00
±0.05b
-1.07
±3.06a
2.89
±0.19b
-69.51
±2.41b
6
47.16
±2.12ab
0.97
±0.15b
-2.83
±0.21ab
2.98
±0.25b
-71.14
±1.39b
All values are means ±SD of three replicates.
a-c
The test values along the same column carrying
different superscripts are significantly different (p< 0.05).
CONCLUSION
In this research work, anthocyanin was extracted from
banana bracts considering two parameters (pH and
solvent concentration) with intra variations. Highly colored
anthocyanins were found at low pH value but it gradually
loses its color during increase in pH values. Extraction rate
was higher at pH 4 in 40% solvent concentration. Hence,
it can be concluded that this study would help people like
to extract anthocyanin from banana bracts as a source of
natural colorants for their food industries to avoid the
carcinogenic effect of synthetic colorants.
ACKNOWLEDGEMENT
The authors are thankful to Teachers and Agro-chemistry
lab technician for helping to complete the work.
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Accepted 11 April 2019
Citation: Ove TA, Kamal MM, Nasim SMNI, Momin MMI,
Mondal SC (2019). Extraction and Quantification of
Anthocyanin from Banana Bracts Using Different pH and
Solvent Concentration. International Journal of Food and
Nutrition Sciences. 4(2): 060-064.
Copyright: © 2019. Kamal et al. This is an open-access
article distributed under the terms of the Creative
Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium,
provided the original author and source are cited.

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