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Anthocyanin from butterfly pea flowers (Clitoria ternatea) by ultrasonic-

assisted extraction

Conference Paper  in  AIP Conference Proceedings · June 2020

DOI: 10.1063/5.0005289

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Anthocyanin from butterfly pea flowers
(Clitoria ternatea) by ultrasonic-assisted
extraction
Cite as: AIP Conference Proceedings 2237, 020069 (2020); https://doi.org/10.1063/5.0005289
Published Online: 02 June 2020

Achmad Qodim Syafa’atullah, Arie Amira, Sonya Hidayati, and Mahfud Mahfud

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AIP Conference Proceedings 2237, 020069 (2020); https://doi.org/10.1063/5.0005289 2237, 020069

© 2020 Author(s).
Anthocyanin from Butterfly pea flowers (Clitoria ternatea)
by Ultrasonic-assisted Extraction
Achmad Qodim Syafa’atullah1,a), Arie Amira 1,b), Sonya Hidayati1,c)
Mahfud Mahfud1,d)
1
Department of Chemical Engineering, Institut Teknologi Sepuluh Nopember, Surabaya, Indonesia

d)
Corresponding Author: mahfud@chem-eng.its.ac.id
a)
achmad.qodims@gmail.com
b)
ariemachda@gmail.com
c)
sonyahidayati126@gmail.com

ABSTRACT. The food industry in Indonesia is now a highly developed industry. To increase the attractiveness of consumers,
producers make innovations including by adding additional coloring substances into food. There are two types of dyes that are
most commonly used, namely synthetic dyes and natural dyes. Synthetic dyes are easy to get, cheap, many choices, but
synthetic dye waste is very dangerous for the environment because it cannot be degraded naturally. Therefore, alternatives
need to be found to save the environment by using natural dyes. One of the natural dyes can be obtained from the flowers of
the plant Butterfly pea (Clitoria ternatea), which contain anthocyanins as natural pigments in the form of blue and red pigments
in acidic conditions. Several studies to extract anthocyanin from Butterfly pea have been conducted conventionally or
maceration. Another alternative to extracting anthocyanin is the ultrasonic-assisted extraction (UAE) method using water as a
solvent has proven to be more efficient. The purpose of this study is to extract butterfly pea flowers by varying the extraction
time, temperature and pH as well as the ratio of bait to solvent. The experiment was carried out in a pyrex beaker glass placed
in an ultrasonic bath, with the experimental variables: extraction time 30-150 minutes, temperature 30-60 oC, pH 3-11, and the
ratio of feed to solvent at 0.02-0.1. Extraction temperature is maintained below 80 oC. The anthocyanin color content was
tested using a UV-Vis spectrophotometer with wavelength absorption of 500-700 nm. The results showed that the highest
anthocyanin concentration was 1,425 g / L at 90 minutes, temperature 60 oC, pH 7, and the ratio of material to solvent was
0.02. From this research it shows that ultrasonic-assisted extraction(UAE) can increase yields higher than conventional
extraction methods.

Keywords: Ultrasonic-assisted extraction; anthocyanin; extraction; Clitoria ternatea, butterfly pea

INTRODUCTION

The food industry in Indonesia is now a highly developed industry. To increase the attractiveness of
consumers, producers make innovations including by adding additional coloring substances into food. There are
two types of dyes that are most commonly used, namely synthetic dyes and natural dyes. Synthetic dyes are easy
to get, cheap, many choices, but synthetic dye waste is very dangerous for the environment because it cannot be
degraded naturally. Therefore, alternatives need to be found to save the environment by using natural dyes.
Lately, the demand for the use of natural dyes from plants for food has increased to replace the use of artificial
dyes. The use of synthetic dyes for food can cause poisoning and carcinogenic [1], [2]. There are many types of
plants that can be used as food coloring. One of them is the butterfly pea or Clitoria ternatea flower, some white,
blue or purple. In Malaysia, the blue color from butterfly pea flower used as food dye in sticky rice. And the
flowers used as vegetable in Kerala (India) and Philippine [3]. The blue color of the butterfly pea flower comes
from anthocyanin, which belongs to their Terrnatin category [4]. Terrnatin is used as a weight loss anti-
inflammatory and other health effect. The peak value of ternatin absorbance lies at wavelengths of 580 nm and
620 nm [5]. Because of the health benefits andJoint
The 14th attractive
Conferencecolors for,2019
on Chemistry the demand for anthocyanin in particular is
AIP Conf. Proc. 2237, 020069-1–020069-6; https://doi.org/10.1063/5.0005289
Published by AIP Publishing. 978-0-7354-1996-4/$30.00

020069-1
 constantly increasing. Anthocyanins are including water-soluble pigment. In the future, it formed as glycosides
of polyhydroxy or polymethoxy derivatives of 2 phenilbenzopyrilium salts, such as aglycones [6].
Clitoria ternatea, also known as the butterfly pea or butterfly pea flower, is a plant species of the Fabaceae
family. Aside from being a coloring agent, this plant is widely used as a memory enhancer, nootropic, anti-stress,
antidepressant, anticonvulsant, tranquilizing and sedative agents. [7]. They are grown as ornamental plants that
propagate on fences and can be found in shrub in dry soil. This plant grows at an altitude of 700 m above sea
level. The flowers axillary, soltary or 2 together, resupiwy, bright bashoate, large, and showy, bright blue [8].
The extraction of dyes from butterfly pea flowers can be done by several methods. The usual methods for
extraction, that are maceration, soxhletation, and reflux methods. This previous method has low efficiency [9],
[10]. At this time, the extraction method with ultrasonic waves or Ultrasonic Assisted Extraction (UAE) was
developed. The UAE method is an alternative method developed to optimize the extraction process. Therefore, in
this study, the extraction of natural dyes from butterfly pea flowers using the UAE method and the factors affecting
extraction were analyzed to be used as a reference for extraction of butterfly pea anthocyanin.

MATERIALS AND METHODS

Materials
The main materials for experiment is Fresh flowers Butterfly peas (Clitoria ternatea) that obtained from mini
garden of Chemical Engineering Department, Sepuluh Nopember. The Chemicals such as citric acid and
ammonium hydroxide (analytical grade PT. Brattaco) used in the experimental work were obtained from local
stores in Surabaya..

Ultrasonic Assisted Extraction Method

A schematic experimental of Ultrasonic Assisted Extraction (UAE) is shown in fig. 1 which is composed of
an ultrasonic cleaning bath with indicator temperature. The spesification of ultrasonic cleaning bath used for this
experiment is KRISBOW model KW1801033, voltage 240 V/50 Hz, maximum power 100 W, maximum
frequency 40 kHz, and tank capacity 2.8 liter. The dimensions of inner tank UAE is length of 23.5 cm, the width
of 13.5 cm, and the height of 10 cm. The dimensions of outer UAE is length of 26.5 cm, the width of 16.5 cm,
and the height of 24 cm.

FIGURE 1. A schematic experimental of ultrasonic-assisted extraction (UAE)

Five plant material to solvent ratio (F/S) of Clitoria ternatea flower samples (0.02, 0.04, 0.06, 0.08 and 0.10
g ml-1) were placed in a beaker glass (200 ml). The beaker was setup on tank with water placed around beaker.
The ultrasonic was operated at maximum frequency for a period time (30, 60, 90, 120, and 150 minutes). The
variables of pH which experimented were 3, 5, 7, 9, and 11. After extraction, the flower taken out, and the extract
solution filtered via Whatman No. 1 filter paper, then take in small bottle to store before scanning UV-Vis
spectrophotometry. Calculation of anthocyanin content : The amount of anthocyanin of butterfly pea can be
calculated by formula:
γ = (A.M.F)/(m.ε.l)
where γ means mass concentration of anthocyanin, A is absorbance, M is molar mass of anthocyanin, 500.8 gmol-
1
, F is dilution ratio of butterfly pea flower, V is volume of extraction solution, m is mass of butterfly pea, ε is
attenuation coefficient, 27300 lmol-1cm-1, and the width of cuvette, 1 cm.

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 RESULT AND DISCUSSION
Ultrasonic-assisted extraction is the renewable technology which is environmentally friendly. When the
solvent is irradiated with ultrasonic waves, the micro bubble emerges. It is due to ultrasonic waves has destructive
character that it can destruct the plant. This condition makes surface tension of solid and liquid decrease. So, there
are mass transfer of solid content and the solvent. The solute can be taken out and extract by solvent [11]. UAE
is suitable to extract the thermo labile compound, such as anthocyanin. Beside reducing time, it is used to avoid
exposure to high temperature. However, there is a deficiency of UAE, need more cost for equipment and still too
hard to apply this method in industry [12]. The previous study, in extraction from different plant materials using
ultrasonic shows that there is an increasing 13-100% efficiency of dye extraction from various plant. It is caused
by using ultrasonic [10], [13].

1. Effect of temperature extraction

1.6
Concentration of anthocyanin (g/l)

1.4

1.2

1
60 C
0.8
45 C
0.6
30 C
0.4

0.2

0
0 20 40 60 80 100 120 140 160
Time (min)

FIGURE 2. Time effect of anthocyanin butterfly pea extraction at 300C, at 450C, and at 600C

Four grams of fresh butterfly pea were soaked by aquadest in a ratio 0.02, and anthocyanin was extracted by
ultrasound at 300C,450C, 600C, 30 min, 60 min, 90 min, 120 min, and 150 min, respectively. From the results, it
is observed that the anthocyanin concentration is increase with long time from 30 to 150 min (Fig. 2). This is due
to the fact that the longer UAE time which the wave can cause the rupture of tissue, it will remove the solute and
anthocyanin produced increasingly. This phenomenon occurs because diffusion of the target compound from the
material matrix into the solvent increases with the extraction time to level certain. A graph that begins to decline
indicates that the binding capacity the solvent to extract has reached its maximum limit [14].
Temperature is one of the key parameters that affect the concentration of anthocyanin from butterfly pea.
Temperature influence the concentration anthocyanin extract from butterfly pea. As shown in figure 2, it is
observed that the concentration of anthocyanin is increased with increasing temperature from 300C to 600C. From
the peak, the highest peak which means the highest content anthocyanin is at temperature 600C. This is due to the
fact that increasing temperature of solvent make solvent diffusifity increase. So, it can easily get in and extract
the solute [15].
When above 800C, butterfly pea flowers were blackish shade which due to organic source and anthocyanin
become unstable. And there is brown precipitate at higher temperature, due to the absence of o-dihidroxy phenolic
compounds and polyphenoloxidase and formed chalcone which make colorless [16][1]. From the result, with
increasing temperature, it can make shorter time to get highest peak. These data suggest that applying higher
temperature maybe the most effective way to extract anthocyanin from Clitoria ternatea flower using UAE.
However, a higher temperature may lead to decrease the anthocyanin concentration because of thermal
degradation of the anthocyanin. It can be concluded that 600C is the best temperature extraction of anthocyanin.

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 2. Effect of plant material to solvent ratio

Concentration of anthocyanin
3.5
3
2.5

(g/l)
2
1.5
1
0.5
0
0 0.02 0.04 0.06 0.08 0.1
Feed to solvent ratio

FIGURE 3. Effect of feed to solvent ratio of anthocyanin butterfly pea extraction ( 600C and 90 min)

From Fig. 2, it can be concluded that highest concentration of anthocyanin from butterfly pea is at 600C and
90 min. This condition will be used to observe the effect of feed to solvent ratio. Feed to solvent ratio is one of
the important parameters which influence the extraction of anthocyanin from butterfly pea flower. The effect of
feed to solvent was carried out in range 0.02 to 0.1. As shown in fig. 3, it is observed that concentration of
anthocyanin increased linearly with increasing feed to solvent ratio. At ratio of 0.08 to 0.1, the anthocyanin
concentration starts stagnant. It caused of solvent has limit to extract the matrix of flower that is anthocyanin. It
can be concluded that the solvent saturated [17].
Fig. 3 shows that the greater feed to solvent ratio, the anthocyanin content of butterfly pea flower got big [16].
It depends on the amount of flower. If the weight of flower increased, the level of anthocyanin also increased.
When feed to solvent ratio arise, amount of water is less, the flower might have not extracted optimum. It caused
of increasing anthocyanin concentration extract produced is not linearly with the increasing of flower mass. So,
yield of anthocyanin must be calculated to observe the optimum extraction.

3. Effect of pH

1.6

1.4
Concentrof anthoation

1.2
anthocyanin (g/l)

0.8

0.6

0.4

0.2

0
0 2 4 6 8 10 12
pH

FIGURE 4. Effect of pH of anthocyanin butterfly pea extraction (600C, 90 min, FS 0.02)

020069-4
 FIGURE 5. Changes effect of pH in color of Clitoria ternatea flower extract

Various pH in this experiment are 3.15, 5.09, 7.01, 9.03, and 11.92. In acidic extraction, solvent added by
citric acid to reach the acid condition. And also, to reach base condition, solvent added by ammonium hydroxide.
Fig. 4 shows that increasing of the amount of anthocyanin from butterfly pea with increase of pH. However, above
pH of 7, there is a decreasing line which means there is decreasing of anthocyanin amount. The optimum
anthocyanin level results are obtained at pH butterfly pea flower extract approaching normal which is equal to
1.425 g/l. Previous research explains that anthocyanin concentration increases with increasing of pH. However,
anthocyanin stable at low pH and become less stable when exposed to heat. Dye extraction of anthocyanin from
butterfly pea at slightly acidic is the highest concentration extract got [18][16],[19].
Fig. 5 shows that there are color changes of anthocyanin in pH effect. At lower pH, the color of blue
anthocyanin becomes light. This is due to anthocyanin change to flavium cation which give red color [17]. The
opposite, the color blue anthocyanin becomes colorless at alkaline condition. This is due to degraded of
anthocyanin which base carbinol and quinodal dominates of flavium cation [16]. So, the optimum blue
anthocyanin concentration is at pH normal.

Acknowledgement
The authors are grateful for the financial support provided by a grant from the Director of Research and
Community Service (DRPM) of the Indonesian Ministry of Research, Technology and Higher Education through
the Master Research scheme.

Conclusions

Anthocyanin extraction from fresh butterfly pea flower by using ultrasonic with 4 variables, that are time
extraction (30-150 min), temperature extraction (30-60 oC), ratio of feed to solvent (0.02-0.1), and pH (3-11).
From the experimental results shows those parameters affect the absorbance of anthocyanin and concentration of
anthocyanin from butterfly pea. Extraction at 60 oC using ratio feed to solvent 0.02. The optimum time for
anthocyanin extraction from butterfly pea flower at 90 minutes. It can make extraction more effective in cost. And
the optimum pH condition to extract of anthocyanin is pH normal (pH 7). Concentration of anthocyanin using this
optimum condition is 1.425 g/l. It can be concluded that extraction of anthocyanin from butterfly pea using
ultrasonic more effective than conventional extraction.

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