18 Optimization of anthocyanin extraction conditions for
Hibiscus rosa-sinesis and Clitoria ternatea using response surface methodology May Suet Seng1, Choon Yoong Cheok*1
1Department of Chemical and Petroleum Engineering, Faculty of Engineering, Technology and Built Environment, UCSI University, 56000 Cheras, Kuala Lumpur, Malaysia.
* Email of corresponding author: cheokcy@ucsiuniversity.edu.my Keywords: Hibiscus rosa-sinensis; Clitoria ternatea; Box–Behnken design
Abstract
This study aimed to investigate the optimum anthocyanin extraction conditions for Hibiscus rosa-sinensis and Clitoria ternatea. Factors including extraction time (10, 35, and 60 min), acid concentration (0, 1, and 2%), and solid to solvent ratio (0.01, 0.03, and 0.05 g/mL) were optimised. Response surface methodology’s Box–Behnken design was employed to optimize the conditions for the maximum response of total monomeric anthocyanin (TMA) based on 15 experiment runs. TMA was determined using pH-differential method and result was expressed in milligram cynadin-3-glucoside per gram fresh weight (mg cy-3-glu/g). Result showed that the optimum extraction conditions to obtain highest TMA value from Hibiscus rosa-sinensis were using 0.36% acetic acid concentration and 0.01 g/mL solid to solvent ratio for 60 min extraction time. Meanwhile, 15 min extraction time, 0.28% lactic acid concentration and 0.0151 g/mL solid to solvent ratio has been found as the optimum extraction conditions for anthocyanins recovery from Clitoria ternatea .
Introduction
Red flowers of Hibiscus rosa-sinesis contain various type of antho- cyanins and flavonoids [1] which are responsible for the red hue pigment and are widely used as the natural colorant. The flowers have been reported having anti-bacterial, anti-oxidant and anti-can- cer activities [2]. Besides, it has been reported that consuming flower part of Hibiscus rosa-sinesis could improve hair growth [3].
Clitoria ternatea is a plant that belongs to the family of Fabaceae where it is also placed in the Papilionaceae family that exhibits con- centrated cobalt blue color. It is also known as butterfly pea in Eng- lish. It is an evergreen twinning herd that can be found abundantly in the South-eastern Asia regions [4]. Butterfly pea can appears in two varieties of colors, which are petals with blue or white colors.
Clitoria ternatea has many uses in different field of industries rang- ing from food to pharmaceutical industries. Both flowers are antho- cyanins rich and proven to have many applications.
The extraction conditions of the anthocyanin from plants can be op- timized using response surface methodology (RSM). Different ex- perimental design and statistical analysis can be used to perform RSM using different software. The extraction conditions to be opti- mized depends on the requirements or objectives of the experiment.
Table 1 listed the optimization of anthocyanin extraction from differ- ent material using different RSM design and software. Box- behnken, central composite and full factorial designs are commonly used RSM for this purpose. However, Box-behnken was more pop- ular selected because of lesser number of experiment runs required.
For a 3 factors – 3 levels design, full factorial design requires 27 runs and central composite design requires 20 runs, unlike Box- behnken, it requires only 15 runs. With lesser number of experiment runs, it saves time and cost. This can be explained as Box-behnken design avoids the treatment combinations that are extreme in terms
of considering the star points and corner points. The treatment com- bination Box-behnken design are at the midpoints of edges of the process space and in the center [5].
Table 1 RSM used to optimize anthocyanin extraction condition in previous studies
This study is designed to evaluate the optimum extraction time, acid concentration, and solid to solvent ratio on recovery of anthocyanin from Hibiscus rosa-sinesis and Clitoria ternatea using Box- Behnken design.
Research Methods
50% ethanol aqueous solvents acidified with acetic and lactic acid, respectively, were used to investigate the optimum extraction con- ditions for Hibiscus rosa-sinesis and Clitoria ternatea. Minitab’s (Version 14) response surface methodology (RSM) Box-Behnken model for three independent variables of extraction time (10, 35, and 60 min), acid concentration (0, 1, and 2%), and solid to solvent
19 ratio (0.01, 0.03, and 0.05 g/mL) with a total of 15 experimental runs
were employed [6,7,8,9].
The independent variables selected for this study were extraction time, acid concentration and solid to solvent ratio at three different levels as listed in Table 2.
Table 2 Independent variables and levels of Box-Behnken design
Anthocyanins content (total monomeric anthocyanins, TMA) of the extract was response variable and quantified using pH-differential method. TMA value was expressed in milligram of cyanidin-3-glu- coside per gram of flesh flower (mg cy-3-glu/g).
Results and Discussion
The experimental design matrix was obtained using Minitab soft- ware and results are displayed in Table 3.
. Table 3 Box–Behnken design matrix with independent variables and measured response (TMA) for Hibiscus rosa-sinesis and Clito- ria ternatea
Hibiscus rosa-
sinesis Clitoria ter- natea Run Extrac-
tion time (min)
Acid concen-
tration (%)
Solid to solvent ratio (g/mL)
TMA (mg cy-3-glu/g)
1 35 2 0.05 0.076 ± 0.002 0.069 ± 0.002
2 35 1 0.03 0.293 ± 0.003 0.121 ± 0.002
3 10 0 0.03 0.270 ± 0.001 0.152 ± 0.003
4 10 2 0.03 0.294 ± 0.005 0.128 ± 0.002
5 60 1 0.05 0.083 ± 0.002 0.069 ± 0.002
6 10 1 0.01 0.892 ± 0.003 0.165 ± 0.006
7 35 2 0.01 0.977 ± 0.005 0.150 ± 0.013
8 35 1 0.03 0.242 ± 0.002 0.116 ± 0.002
9 60 0 0.03 0.231 ± 0.003 0.139 ± 0.003
10 35 0 0.01 0.987 ± 0.006 0.168 ± 0.012
11 35 1 0.03 0.259 ± 0.003 0.130 ± 0.002
12 10 1 0.05 0.117 ± 0.000 0.077 ± 0.001
13 35 0 0.05 0.072 ± 0.002 0.082 ± 0.007
14 60 1 0.01 1.051 ± 0.009 0.141 ± 0.010
15 60 2 0.03 0.216 ± 0.004 0.108 ± 0.002
Mean ± SD (standard deviations) of duplicate analysis (n= 6). TMA= Total mono- meric anthocyanin; mg cy-3-glu= mg cyanidin-3-glucoside
The best extraction conditions for Hibiscus rosa-sinesis was 60 min with 1 % acetic acid concentration (1 mL of acetic acid solution with 99 mL of ethanol aqueous solution) and 0.01 g/mL solid to solvent ratio (1 gram in 100 mL of solution) which obtained the highest TMA of 1.051 ± 0.009 mg cy-3-glu/g (Table 1). The optimum conditions for Clitoria ternatea to attain the highest anthocyanins of 0.168 ±
0.012 mg cy-3-glu/g were 35 min without the addition of organic acid (100 mL of ethanol aqueous solution) and 0.01 g/mL solid to solvent ratio (1 gram in 100 mL of solution).
The predicted models for Hibiscus rosa-sinesis (Y1) and Clitoria ter- natea (Y2) were as the following:
Where Y1 is the TMA yield (mg cy-3-glu/g FW) for Hibiscus rosa- sinesis, Y2 is the TMA yield (mg cy-3-glu/g FW) for Clitoria ternatea, 1.4212 and 0.1992 are the intercept, A, B, C are the linear coeffi- cients, AA, BB and CC are squared coefficient, AB, AC and BC are interaction coefficient, A is the extraction time, B is the acid concen- tration and C is the solid to solvent ratio.
TABLE 4 Statistical analysis and model fitting
(a) Hibiscus rosa-sinesis using 50 % ethanol aqueous extraction solution acidified with acetic acid
(b) Clitoria ternatea using 50 % ethanol aqueous extraction solution acidified with lactic acid
Table 4 presents the summary of ANOVA (Analysis of Variance) for both flowers, both models were 0.000 which were highly significant (p<0.01) with the evident of high Fisher value (F value) from the Fisher’s F-test and low Probalility value (P value). The F value is used to prove the efficency of model, with high F value indicates high model efficiency. Moreover, the lack of fit shows the variation
20 due to the model inadequacy [10]. The lack of fit of Hibiscus rosa-
sinesis was 0.208 and Clitoria ternatea was 0.683 which was not significant (p>0.05). The coefficient of determination (R2) is calcu- lated using the following equation:
𝑅2= 1 −𝑅𝑆𝑆 𝑇𝑆𝑆
Where RSS is residual sum of square and TSS is total sum of square. It is interpreted based on the proportion of the variability in the data and the significance of the model. The R2 values of Hibis- cus rosa-sinesis and Clitoria ternatea were 0.9950 and 0.9883 re- spectively. This implied that more than 95% of the experimental data can be explained by the model and the model has high effi- ciency as well as its significance level.
According to the model, solid to solvent (p value for both model is 0) was found to be the most significant factor (p<0.01) on the TMA yields, followed by acid concentration and extraction time which showed less significant (p>0.05 for Hibiscus rosa-sinesis and p<0.05 for Clitoria ternatea) for both flowers. Besides, the quadratic terms (A2, B2 and C2) were not significant (p>0.05) except solid to solvent ratio (C2) that was significant (p<0.05) for both flowers.
Among the interaction terms (AB, AC and BC) for hibiscus, not sig- nificantly different (p>0.05) between these interactions except the interaction between extraction time and solid to solvent ratio (AC) although the p value is 0.076 that was slightly more than 0.05 but it was lesser compared to the other two interactions. As for the inter- action terms of Clitoria ternatea, it was observed that not significant (p>0.05).
Figure 1(a) shows that as the extraction time increased from 10 min to 60 min, TMA value increased slightly. But, the relationship be- tween TMA and solid to solvent ratio was vice versa. As the solid to solvent ratio increased from 0.01 to 0.05 g/mL, TMA decreased gradually from 1.0 to 0 mg cy-3-glu/g. The extraction time increased from 10 to 60 min, TMA increased from 0.24 mg cy-3-glu/g to about 0.26 mg cy-3-glu/g (Figure 1(b)). Additionally, the TMA increased with the increased of acid concentration up to certain limit, after the limit, the TMA decreased gradually. This is due to high acid con- centration degrades the anthocyanins and hence, reduces the TMA [5]. TMA decreased from 1.0 to 0 mg cy-3-glu/g when the solid to solvent ratio increased (Figure 1(c)). The effect of solid to solvent ratio and acid concentration was not significant (p>0.05) to the TMA.
However, solid to solvent ratio has significance effect on TMA.The interaction of both factors with TMA yields was not significant (p=0.076, p>0.05) but it can be seen that solid to solvent ratio showed more effect on the TMA yields as solid to solvent ratio demonstrated significant value in this model based on Table 4(a).
It showed that TMA yields increased when extraction time in- creased while solid to solvent ratio decreased. This phenomena might due to longer extraction time allows more interaction between the solid with the acidified solution, but as the solid to solvent ratio increase, it reduces the contact of the solid with solvent which lead to reduction of anthocyanin yields due to the increased of solid in
the solvent [6]. According to Zou et al. [7], a larger solvent volume enhanced the TMA yields due to the solvent was able to dissolve the constituent more effectively. In agreement to it, lower solid to solvent ratio is preferred as lower solid amount in a fixed volume of solvent increases the contact of solid with solvent, where this in- creases the extraction of anthocyanins. Thus, the maximum TMA yields can be attained with low solid to solvent ratio and high ex- traction time.
Figure 1 Surface diagrams of effects of (a) extraction time and solid to solvent ratio, (b) extraction time and acetic acid concentra- tion, and (c) acetic acid concentration and solid to solvent ratio, on
TMA for Hibiscus rosa-sinesis
(a)
(b)
(c)
21 Figure 1(b) demonstrates the relationship of extraction time and
acid concentration with TMA yields. The extraction time increased from 10 min to 60 min, the TMA yields increased from 0.24 mg cy- 3-glu/g FW to about 0.26 mg cy-3-glu/g FW. Additionally, the TMA yields increased with the increased of acid concentration up to cer- tain limit, after the limit, the TMA yields decreased gradually. The interaction between these two factors with TMA yields was not sig- nificant (p>0.05). The correlation between these two factors with TMA yields might due to increase of acetic acid concentration which favours the extraction of anthocyanin. However, higher acetic acid concentration might further degrade the anthocyanins and reduces the yields [11]. Thus, the maximum TMA yields can be acquired at optimum acid concentration with high extraction time.
Figure 1(c) displays the relationship of acid concentration and solid to solvent ratio with TMA yields. The TMA yields showed very slight changed with the acid concentration increased. The TMA yields de- creased from 1.0 mg cy-3-glu/g FW to 0 mg cy-3-glu/g FW when the solid to solvent ratio increased. The relationship between these two factors with TMA yields demonstrated the TMA yields in- creased with the acid concentration increased slightly and de- creased of solid to solvent ratio. The effect of solid to solvent ratio and acid concentration was not significant (p=0.877, p>0.05) to the TMA yields, but the interaction between them was mainly the effect of solid to solvent ratio and due to this factor was very significant in the model based on Table 4(a). Since the main effect on the TMA yields for this set of comparison is mostly the factor of solid to sol- vent ratio, thus it played a significant role in affecting the TMA yields.
The decreased of solid to solvent ratio generally decreases the solid amount in the fixed solvent volume and at the same time in- creases the solvent volume. With an increase of solvent volume enhances the solubitliy of solid in the solvent and better interaction of solvent with solid. Thus, it improves the TMA yields extracted from the flower [7].
Solid to solvent ratio posed significant impact (p<0.05) on the model compared to the other two factors as illustrated in Table 4 (a) and the decreased in solid to solvent ratio increased the TMA yields.
Thus, it is recommended that low solid to solvent ratio to be used for the extraction of TMA from Hibiscus rosa-sinesis. Besides, an optimum acetic acid concentration is recommended for extraction as too high acid concentration will further hydrolyse the acylated anthocyanins or too low acid concentration results in the reduced ability to extracted the anthocyanin from the flower will causes the reduce the TMA yields. Furthermore, long extraction time will be recommended due to long extraction time allows the more antho- cyanins to be extracted.
Figure 2 (a) shows that as the extraction time increased from 10 to 60 min, TMA decreased slightly. Similarly, the solid to solvent ratio increased from 0.01 to 0.05 g/mL, the TMA decreased from 0.15 to 0.06 mg cy-3-glu/g. No significant interaction between extraction time and solid to solvent ratio to TMA. This indicated that TMA in- creased when extraction time and solid to solvent ratio decreased.
Figure 2 (b) demonstrates TMA increased with the extraction time reduced from 60 to 10 min while acid concentration reduced from 2 % to 0 %. No interaction between extraction time and acid con- centration on TMA indicated that TMA increased in reducing extrac- tion time and acid concentration. This could be explained as high lactic acid concentration might cause more hydrolysis of acylated moities [12]. Increased of extraction time allowed more acid to react
with anthocyanins and resulted even more hydrolysis of acyl moie- ties which led to TMA reduction. Figure 2(c) illustrates TMA in- creased with the decreased of lactic acid concentration from 2 % to 0 % and decreased of solid to solvent ratio from 0.05 to 0.01 g/mL.
As a result, the maximum TMA can be attained with low lactic acid concentration and solid to solvent ratio. This might also due to high solid to solvent ratio reduces the contact of solid in fixed amount of solvent and a lower volume of solvent reduces the solubility of solid in solvent which reduces the extraction efficiency, while high con- centration of acid further lead to the hydrolysis of acylated anthocy- anins.
Figure 2 Surface diagrams of effects of (a) extraction time and solid to solvent ratio, (b) extraction time and lactic acid concentra- tion, and (c) lactic acid concentration and solid to solvent ratio, on
TMA for Clitoria ternatea
(a)
(b)
(c)
22 From the RSM analysis, the optimum extraction conditions of an-
thocyanins from Hibiscus rosa-sinesis were 60 min extraction time, of, 0.36% acetic acid concentration and 0.01 g/mL solid to solvent ratio. While for Clitoria ternatea, the optimum extraction conditions were 15 min extraction time, 0.28% lactic acid concentration and 0.0151 g/mL solid to solvent ratio. Experiment was conducted in duplicate to verify these optimum conditions. The results obtained were 0.95 ± 0.00 mg cy-3-glu/g FW for Hibiscus rosa-sinesis and 0.15 ± 0.00 mg cy-3-glu/g FW for Clitoria ternatea. The percent de- viation between the experimental data and actual software pre- dicted results were calculated. The percent deviation calculated for Hibiscus rosa-sinesis was 10.0% and Clitoria ternatea was 9.4 %.
These percent deviation were considered acceptable as it was about 10% [13]. Thus, this validation study confirmed that the mod- els fitted the experimental data well for the TMA yields and these optimum extraction conditions are reliable for both flowers studied.
Conclusion
The optimum anthocyanin extraction conditions for Hibiscus rosa- sinensis and Clitoria ternatea were 60 min extraction time, of, 0.36%
acetic acid concentration, 0.01 g/mL solid to solvent ratio and 15 min extraction time, 0.28% lactic acid concentration and 0.0151 g/mL solid to solvent ratio, respectively. RSM demonstrated that solid to solvent ratio has significant effect on TMA.
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