SUPPLEMENTARY MATERIAL
Ultrasonic extraction of anthocyanin from Clitoria ternatea flowers using response surface
methodology
Fui Chin Chong* and Xian Fu Gwee
Faculty of Chemical and Natural Resources Engineering, Universiti Malaysia Pahang,
Lebuhraya Tun Razak, 26300 Kuantan, Pahang, Malaysia
*Corresponding author E-mail: fcchong@ump.edu.my
The ultrasonic extraction (UE) method of anthocyanin from Clitoria ternatea flowers using
response surface methodology was performed in this study. By using response surface
methodology (RSM), the objective is to optimise the extraction yield of anthocyanin from
Clitoria ternatea which is influenced by various factors, including the extraction temperature,
time, ratio of solvent to solid and ultrasonic power. The empirical model was investigated by
performing first level optimization in two-level factorial design with Design Expert 7 software.
In comparison with the conventional solvent extraction (SE), ultrasonic extraction (UE) showed
a 246.48 % better extraction yield and produced an anthocyanin extract with a radical scavenging
activity of 68.48% at the optimized factors of 50°C, 150 min, 15 ml/g and 240 W.
Keywords: Clitoria ternatea; ultrasonic extraction; anthocyanin; antioxidant activity; response
surface methodology
A Experimental
A.1 Plant material and chemicals
Fresh Clitoria ternatea flowers were obtained through self-planting from Balik Pulau, Penang,
Malaysia. Only individual Clitoria ternatea flower that was free of defects and diseases was
chosen for this study. Fresh collection of blue pea flowers was washed with distilled water and
stored in -25°C freezer and used for extraction within a month. Only the petals of fresh Clitoria
ternatea flowers were used in extraction process. They were cut into smaller pieces (average size
of 0.5 cm x 0.3 cm) using a blender (MX-800S, Panasonics, Selangor, Malaysia) before usage.
Only a single batch collection of flowers was used for extraction purpose to eliminate
inconsistency of flowers batches. In addition, the wet flowers sample without any oven drying
was used for extraction as it was interesting from technological point of view. This was due to
that the sample did not require previous drying of the source. In addition, wet flowers sample
was easier to separate the extracts from exhausted vegetal source compared to fine ground
particles. This provided an edge against the long and conventional method of oven-drying and
maceration of vegetal source sample before extraction process.
Potassium chloride (KCl) and sodium acetate (NaC2H3O2) were purchased from Merck
(Selangor, Malaysia). Acetic acid glacial, L-Ascorbic acid of analytical reagent grade and
hydrochloric acid were purchased from Fisher Scientific (Selangor, Malaysia). 2,2-diphenyl-2picrylhydrazyl (DPPH) was purchased from Sigma-Aldrich (Kuala Lumpur, Malaysia). 95%
ethanol was purchased from Copens Scientific (Selangor, Malaysia).
A.2 Ultrasonic Extraction
Ultrasonic extraction (UE) was done according to the modified method of Chen et al. (2007).
Distilled water was used as solvent. Ultrasonic bench top cleaner machine (CP1800, Crest
Ultrasonics, New York, USA) with power from 96 to 240 W was used for ultrasonic extraction.
The UE variables were temperature (30-50°C), time (30-150 min), ratio of solvent to solid (2-15
ml/g) and ultrasonic power (96-240 W). The temperature was controlled and maintained at
targeted temperature by periodically adding ice into and removing water from the ultrasonic
machine. Anthocyanin extract was filtered through Whatman No. 1 paper twice where the
residue was gathered and extracted again under the same conditions. The anthocyanin extract
was determined using UV-VIS spectrophotometer (U1800, Hitachi HighTech, Kuala Lumpur,
Malaysia). The colour change of sample during the ultrasonic extraction was observed in Figure
S1.
A.3 Conventional Solvent Extraction (SE)
This was done using the modified version of the process described by Fuleki and Francis (1968).
One gram of sample was added with 15 mL of 1.5 M HCl–95% ethanol. The SE variables were
set to be equivalent to the optimised UE variables, which were temperature (50°C), time (150
min), ratio of solvent to solid (15 ml/g). Solvent extraction (SE) was carried out in a water bath
machine (ONE 14, Memmert, Schwabach, Germany).
A.4 Experimental design for UE
The design of experiment (DOE) for UE was done according to two-level factorial design in
Design Expert 7 software. However, only the first level known as screening test (ST) of the twolevel factorial design was performed in this study. Extraction variables were extraction
temperature (°C), extraction time (min), ratio of liquor to solid (ml/g) and sonication power (W).
Extraction variables were input according to their upper and lower limit and numerical or
categorical factor for the ST as shown in Table S1. Range of parameters values was entered into
Design Expert 7 software which was obtained from literature review for the trial runs for ST. To
generate the DOE for ST, two level factorial design was chosen where 4 factors were chosen
with 8 runs and 3 replicates. 8 runs (half design) were chosen instead of 16 runs (full design) due
to the time and resource constraint in conducting 64 runs to 24 runs for 3 replicates each. Then,
the extraction variables were entered according to their label, unit, type and upper and lower
limit. All were numerical factor except for sonication power at categorical factor as explained
earlier. Next, the number of response was selected which was only anthocyanins content with its
label and unit. Lastly, the DOE for ST was generated and presented in Table S2.
A.5 Determination of total anthocyanin content (TAC)
TAC determination was performed according to the modified method of Kerio et al. (2012). As
shown in Figure S2, 200 µl of ultrasonic extraction (anthocyanin extracts) sample was dissolved
in 1.8 ml of 0.025 M potassium chloride (KCl) buffer at pH 1.0 and 0.4 M sodium acetate
(NaC2H3O2) buffer at pH 4.5 each with a pre-determined dilution factor of 10. This is known as
the pH differential method. Blank reading for measurement was taken using deionized water.
Then, measurement by UV-VIS spectrophotometer on absorbance at 520 nm and 700 nm was
done in triplicate. The absorbance (A) of the diluted sample was calculated as in Eq. (3) and Eq.
(4).
A = (A520 – A700)pH1.0 – (A520-A700)pH4.5
Anthocyanin content (mg/L) =
(3)
A×MW×DF×1000
(4)
e ×1
where MW (molecular weight) of anthocyanin equivalent, delphindin-3-glucoside = 500.8, DF
(dilution factor) = 10, e = 29,000 molar absorbance of delphinidin-3-glucoside. The anthocyanin
content was calculated from delphindin-3-glucoside equivalent.
A.6 Comparison of anthocyanin content between UE and SE
The comparison of yield between ultrasonic extraction and conventional solvent extraction with
reference to anthocyanin content of both extractions was calculated by Eq. (5).
Yield comparison, % =
ACOUE -ACOCSE
ACOCSE
× 100%
(5)
where ACOUE (mg/g) is the anthocyanin content of ultrasonic extraction and ACOCSE (mg/g)
is the anthocyanin content of conventional solvent extraction.
A.7 Comparison of ST and validation test (VT) of anthocyanin content
The comparison was done in term of difference between both tests. Validation test (VT) was
performed according to the optimized experimental variables value. The difference was
calculated by Eq. (6). It was considered accurate shall the difference was less than 10%.
Difference, % =
ST - VT
VT
× 100%
(6)
A.8 DPPH radical-scavenging activity assay
The DPPH radical-scavenging activity was estimated by the modified method of Bondet et al.
(1997). Aliquots (0.5 ml) of 0 (control), 25, 50, 100, 142.86, 333.33 and 1000 µg/ml of ascorbic
acid dissolved in 95% ethanol were added to 2.5 ml of 0.2 mM DPPH solution in 95 % ethanol.
Anthocyanin extracts conducted in the DPPH assay were obtained from the validation test of
anthocyanin extracts. Concentrations of anthocyanin extracted of 5.45, 9.23, 14.01, 23.41 and
73.62 µg/ml were used to replace ascorbic acid used earlier for DPPH assay. The absorbance at
517 nm of samples was measured after 30 min of incubation at room temperature in the dark.
Measurements were done in triplicates. The percentage of DPPH radical-scavenging activity was
calculated as in Eq. (7).
DPPH, % = [1-
(A-B)
A0
] ×100%
(7)
Where A = absorbance of sample with DPPH solution in ethanol with DPPH solution in ethanol
as blank, B = absorbance of 0.5 ml of extracts + 2.5 ml of ethanol with ethanol as blank, A0 =
absorbance of control.
References
Bondet, V., Brand-Williams, W., & Berset, C. (1997). Kinetics and mechanism of antioxidant
activity using the DPPH free radical method. LWT-Food Science and Technology, 30, 609–
615.
Chen, F., Sun, Y.Z., Zhao, G.H., Liao, X.J., Hu, X.S., Wu, J.H., & Wang, Z.F. (2007).
Optimization of ultrasonic-assisted extraction of anthocyanin in red raspberries and
identification in extract using high-performance liquid chromatography-mass spectrometry.
Ultrasonics Sonochemistry, 14, 767-778.
Fuleki, T., & Francis, F.J. (1968). Quantitative methods for anthocyanin. 3. Purification of
cranberry anthocyanin. Journal of Food Science, 33, 266–274.
Kerio, L.C., Wachira, F.N., Wanyoko, J.K., & Rotich, M.K. (2012). Characterization of
anthocyanin in Kenyan teas: Extraction and identification. Food Chemistry, 131, 31-38.
Table S1.
Screening test results
Run
A
B
C
D
Anthocyanins content, Y (mg/g)
1
50
150
2
96
0.028551
2
50
150
15
240
0.935978
3
30
30
15
240
0.144196
4
50
150
15
240
1.125937
5
30
30
15
240
0.147650
6
30
30
15
240
0.147650
7
30
150
2
240
0.092792
8
50
30
2
240
0.124567
9
30
150
2
240
0.077941
10
50
150
15
240
1.124210
11
30
150
15
96
0.138152
12
30
150
2
240
0.107413
13
50
30
2
240
0.124567
14
50
30
2
240
0.133777
15
50
150
2
96
0.031430
16
30
30
2
96
0.107528
17
50
30
15
96
0.141606
18
50
150
2
96
0.025328
19
50
30
15
96
0.145923
20
30
30
2
96
0.101311
21
50
30
15
96
0.149377
22
30
150
15
96
0.138152
23
30
150
15
96
0.143332
24
30
30
2
96
0.108794
A: extraction temperature, B: extraction time, C: ratio of liquor to solid, D: sonication power
Table S2.
ANOVA results for screening test
S
SSb
DFc
MSd
F-value
Prob-F
Me
2.40
7
0.34
224.44
< 0.0001
A
0.29
1
0.29
189.61
< 0.0001
B
0.24
1
0.24
156.13
< 0.0001
C
0.49
1
0.49
318.75
< 0.0001
D
0.38
1
0.38
250.00
< 0.0001
AB
0.26
1
0.26
172.00
< 0.0001
AC
0.35
1
0.35
228.08
< 0.0001
AD
0.39
1
0.39
256.52
< 0.0001
f
0.02
16
0.00
PE
S: Source, SS: Sum of square, DF: Dilution factor, MS: Mean square, M: Model, PE: Pure error
Figure S1.
Color change (left to right) of water solvent during ultrasonic extraction.
Figure S2.
Anthocyanins extracts in KCl buffer (pink solution) and NaC2H3O2 buffer (blue solution).