REGULAR ARTICLE

OPTIMIZATION OF ANTIOXIDANT EXTRACTION FROM KALUMPIT (TERMINALIA

MICROCARPA DECNE) FRUITS

Dennis Marvin Santiago*1, Claire Zubia1, Shekayna Eunice Pacia1, Sheba Mae M. Duque1
Address: Dennis Marvin Santiago, PhD
1
University of the Philippines Los Baños, College of Agriculture and Food Science, Institute of Food Science and Technology, 4031 College,
Laguna, Philippines, +63495018932

*Corresponding author: [email protected]

ABSTRACT

The effects of extraction parameters, including temperature (25 – 80 °C), time (30 – 90 min), solvent to sample (S/S) ratio (10 – 50 mL g-1),
initial pH (3 - 8) and ethanol concentration (20 – 100%), on the % 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of
kalumpit were screened and optimized using 2-level factorial design and Box-Behnken design (BBD) of experiments. Temperature, S/S ratio,
and ethanol concentration exhibited significant effects on the % DPPH radical scavenging activity of kalumpit extract. Response surface models
developed for % DPPH and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging activities of kalumpit fruit extract
adequately fit and were used to determine the optimum extraction conditions. A desirability function approach determined the optimum
conditions for solvent extraction of antioxidants at 80.0 °C, 10 mL g -1 S/S, and 51.66% ethanol concentration. This resulted in a maximum
desirability value of 0.977 and predicted % DPPH and ABTS radical scavenging activities of 66.63 and 82.14, respectively. Validation of the
adequacy of the predictive models showed no significant difference between experimental data and predicted values (p > 0.05), indicating that
the models developed were adequate in describing the relationship between factors and responses.

Keywords: Solvent extraction, 2-level factorial design, Box-Behnken design, DPPH scavenging activity, ABTS scavenging activity

INTRODUCTION

The Philippines has a variety of edible and nutritious fruits from indigenous trees that are good sources of ethnobotanical ingredients (Dela
Cruz, 2012). Traditionally, fruits that contain phytochemicals are being consumed as food and medicine due to the health benefits they bring.
However, despite their potential health benefits, the economic significance of these fruits is not yet fully realized; thus, they remain
underutilized in terms of food processing. These kinds of fruits are usually seasonal and have a short shelf life. Therefore, it is important to
develop processing technologies to fully realize their benefits and extend their availability in the market.
Kalumpit (Terminalia microcarpa Decne.) is one of the endemic, underutilized, and widely distributed fruit trees in the Philippines. It can be
found in low altitude forests (Department of Agriculture of the Philippines, 1996; Langenberger et al., 2009; Coronel, 2011). Ragasa et
al. (2014) reported that its dichloromethane leaf extract contains squalene, lutein and fatty alcohols that have preventive and therapeutic effects
on tumor promotion and regression, and protective effect against inflammatory damage development. Kalumpit fruits can be eaten raw and are
used in making wine, jam, and jellies (Sanchez et al., 1976; Ungson, 2001). It is also rich in ascorbic acid, enzymes, bioflavonoid, chromium,
potassium, magnesium, B vitamins and amino acids (De Leon, 2012). Moreover, Garcia et al. (2005) reported that kalumpit methanolic fruit
extract has antioxidant activity comparable to butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), DL-α tocopherol and bignay
(Antidesma bunius (L) K. Spreng) fruit in terms of lipid peroxidation inhibition and hydroxyl scavenging activity. It was also verified to have
a high total phenolic and flavonoid content that are responsible for its high antioxidant activities (Santiago et al., 2007).
Antioxidants are compounds that can scavenge free-radicals, which if stabilized and accumulated in the body may cause various pathological
conditions such as various types of cancer, atherosclerosis, coronary heart disease, rheumatoid arthritis and other degenerative diseases (Ramis-
Ramos, 2003). Stabilizing free-radicals is done by means of donating a hydrogen atom to the free-radical to decrease its activation energy, and
to prevent it from reacting with other compounds, which in turn produces more free radicals (Pokorny, 2007; Embuscado, 2015; Rodriguez-
Amaya, 2015; Shahidi, 2015). Polyphenols are compounds with significant antioxidant activity. The bioactive polyphenols are anthocyanin,
flavonoid, isoflavone, phenolic acid and lignin, which exhibit high levels of antioxidant activity (Galili and Hovav, 2014). The extraction of
different compounds associated with antioxidant activity may differ through different sources, methods and factors such as the S/S ratio (Yuan
et al., 2015), solvent concentration (Chen et al., 2015), extraction temperature (Belwal et al., 2016), extraction time (Vaji´c et al., 2015) and
initial pH (Sharmila et al., 2016). The extraction of bioactive compounds was reported to be highly influenced by the processing parameters
employed. The efficiency of extraction is a function of the characteristic of both solvent (e.g. concentration and pH) and compound (e.g. particle
size, ratio to solvent), and extraction time and temperature (Gertenbach, 2001; Cacace and Mazza, 2003). The extraction process also
depends on the diffusion coefficient or how fast the compound will dissolve and reach equilibrium concentration in the solvent, which is
influenced by the concentration of the solute, time and temperature (Cacace and Mazza, 2003). Moreover, the denaturation of plant membrane
tissues was earlier reported to occur at 55-75 °C and cannot be increased indefinitely (Schwartzberg and Chao, 1982).
Research on antioxidant extraction were conducted on mangosteen hull (Cheok et al., 2012), blueberry pomace wine (He et al., 2016),
pomegranate husk (Amyrgialakia et al., 2014), and fruits of peach (Mokrani and Madani, 2016), apple (Alberti et al., 2014) and pear (Guan
et al., 2015) to find natural sources to develop food supplements and functional food products (Silva et al., 2007). This research on kalumpit
is vital to establish baseline data prior to the utilization of this crop and maximize its potential. Aside from food products that can be developed
from kalumpit, high value ingredients, such as antioxidants and colorants, can also be extracted for use in various industries.
In order to maximize antioxidant extraction, the optimization of various parameters and conditions that can affect the extraction process should
be performed. The traditional way of screening variables is the “one-variable-at-a time’ concept that has been shown to work well when applied
to process designs, especially in cases where a small number of variables are being studied. However, this may become a problem when applied
to an experiment that needs to consider a large number of variables. This traditional method is time consuming, expensive and does not allow
the understanding of the interactions between the factors considered (Weuster-Botz, 2000). Response surface methodology (RSM) can be a
better alternative due to its efficiency and practicality. It utilizes mathematical and statistical techniques in the creation of models for the
analysis in which the response of concern can be affected by the numbers of known variables. In a way, those variables are used to attain the
goal of optimal results (Montgomery, 2013).
This study fundamentally aimed to optimize the extraction protocol for antioxidants from kalumpit fruit. Specifically, this study intended to
screen factors (temperature, time, S/S ratio, initial pH and ethanol concentration) that significantly affect the antioxidant activity of kalumpit
extract using 2-level factorial experimental design; optimize levels of significant factors that affect DPPH and ABTS radical scavenging
activities using Box-Behnken design of experiment; and validate the optimized extraction parameters generated.

MATERIAL AND METHODS

Sample Preparation

Mature kalumpit fruits were obtained from the Fruit Crops Nursery, Institute of Crop Science, College of Agriculture and Food Science,
University of the Philippines Los Baños. Fruits were immediately washed, cut to obtain the flesh, dried at 50 °C for 72 hours, ground, and
passed through a 40-mesh sieve. Samples were then stored in an amber or foil-covered bottle under freezing temperature until use.

Chemicals

Absolute ACS/USP Grade Ethyl alcohol (PHARMCO-AAPER, Connecticut, USA), 2,2-Diphenyl-1-picrylhydrazyl (DPPH), and 2,2-azino-
bis-3-ethylbenzothiazoline 6-sulfonic acid (ABTS) (Sigma Aldrich Pte. Ltd, Singapore, Singapore) were used in this study.

Screening of Factors for Antioxidant Extraction

A randomized 2-level factorial experimental design was generated using Design Expert version 10.0.3 (Stat-Ease, Inc., Minnesota, USA) to
screen which among the considered factors significantly affect antioxidant extraction from kalumpit. Varying levels of temperature (25, 52.5,
80 °C), extraction time (30, 60, 90 min), S/S ratio (10, 30, 50 mL g-1), pH (3, 5.5, 8) and % ethanol (20, 60, 100%) were considered based on
previous studies of Chew et al. (2011), Vaji´c et al. (2015) and Belwal et al. (2016). Table 1 shows the different factors considered, in uncoded
levels, consisting of 35 experimental points, including 3 replicates of the center points.

Extraction of antioxidants from kalumpit powder was conducted according to Belwal et al. (2016), with minor modifications. Approximately
1 g of kalumpit powder was weighed into 50-mL centrifuge tubes, added with ethanol (varying amounts and concentration), and adjusted to its
corresponding initial pH. The mixture was then thoroughly mixed using a vortex mixer for 2 minutes and incubated in water bath (varying
temperature and time). Samples were then filtered through Whatman no. 2 filter paper and the collected extract was analyzed for its DPPH
radical scavenging activity.

Optimization and Validation of Significant Factors for Antioxidant Extraction

After identifying the significant factors that influence antioxidant extraction, Box-Behnken Design (BBD) was employed to establish the
optimal values of these factors, based on the maximum percentage of DPPH radical scavenging activity of kalumpit fruit extract. Design Expert
version 10.0.3 was used to generate the model and analyze the results of the experiment. Table 2 shows the different factors, in uncoded levels,
to determine the optimal levels of identified significant factors for maximum antioxidant extraction. Evaluation of % DPPH was conducted.
At the same time, an additional assessment by ABTS radical scavenging activities was carried out as a complementary test. The desirability
function of Design Expert version 10.0.3, which is a unique and powerful multi-response (multivariate) optimization tool, was used to
determine the optimum conditions for extraction of kalumpit with optimum of % DPPH and ABTS radical scavenging activities. To validate
the predictive ability and accuracy of the models developed, three replications at optimum conditions were carried out and compared with the
predicted values.

Analysis of Antioxidant Property

The 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay, according to Zhu et al. (2002), was used to determine the radical scavenging activity of
kalumpit extract. Particularly, 1 mM solution of DPPH was prepared in absolute ethanol. A 1 mL aliquot of diluted extract (dilution factor =
30) in 52% ethanol was mixed with 4 mL distilled water and 1 mL of the freshly prepared DPPH ethanolic solution. The mixture was placed
in a dark room for 30 minutes. The absorbance was measured at 517 nm against a blank using UV-VIS spectrophotometer Shimadzu UVmini-
1240 (Kyoto, Japan). All measurements were carried out in triplicate. The % DPPH radical scavenging activity was calculated using equation
1:

𝐴0 − 𝐴𝑡
% DPPH radical scavenging activity = × 100% Equation 1
𝐴0

where A0 represents the absorbance of control reference at t = 0 min while At represents the absorbance of the samples.

The ABTS (2,2-azino-bis-3-ethylbenzothiazoline 6-sulfonic acid) free radical scavenging activity of the extract was measured using the method
described by Re et al. (1999). The ABTS stock solution was prepared by mixing 7 mM ABTS with 2.45 mM potassium persulfate and incubated
for 16 hours in the dark at 30 °C. An aliquot of the stock solution was diluted with methanol to adjust the absorbance to 0.70 ± 0.02 at 734 nm.
A 20 μL aliquot of diluted extract (dilution factor = 30) was added with 980 μL ABTS reagent. The absorbance of the resulting solution was
read at 734 nm using a UV-VIS spectrophotometer Shimadzu UVmini-1240 (Kyoto, Japan) after 1 minute. The antioxidant activity of the
extract was calculated against a calibration curve (0, 5, 10, 15, 20, 25, 30, 35, 40 μM) established with Trolox (R 2 = 0.998), and expressed as
mg Trolox equivalent per 100 g purée. Results were expressed as % ABTS scavenging activity using equation 2:

Absorbance 𝑠𝑎𝑚𝑝𝑙𝑒 Equation 2

Statistical data analysis %ABTS Scavenging Activity = (1 − ) 𝑥 100
Absorbance 𝑏𝑙𝑎𝑛𝑘
All statistical analyses were performed using Design Expert version 10.0.3 software (Stat-Ease, Inc., Minnesota, USA) to analyze the
experimental data and the response surface model for screening and optimization of significant factors affecting % DPPH and ABTS radical
scavenging activities. Design Expert version 10.0.3 software was also used to determine the Analysis of Variance (ANOVA) and coefficient
of determination (R2), and to evaluate the goodness of fit of the model. The validity of the model was determined by comparing the observed
experimental and predicted values for the response variables with t-test analysis using Minitab version 18.1 (Minitab Pty Ltd, Sydney,
Australia).
RESULTS AND DISCUSSION

Screening of Factors for Antioxidant Extraction

A 2-level factorial experimental design, with three-level factor, was employed to screen the extraction factors that significantly affects DPPH
radical scavenging activities of kalumpit extract. Table 1 shows the DPPH radical scavenging activities of kalumpit extracted using different
extraction conditions. The model as described in Equation 3, achieved an R² value of 0.8671, indicating that 86.71 % of the proportion of the
variability in the response can be explained by the model. Moreover, the high value of R² (values > 0.70) is indicative that the model is fitting
the data very well. A standard deviation value of 3.06 apart from the fitted values, which is only 4.62 % of the mean (66.08%), signifies a
precise description of the response. Nevertheless, the model assumption cannot be validated completely through this value, hence the need for
further statistical analyses.

DPPH Scavenging Activity = +66.08 +1.96A +0.40B -2.15C -0.11D -1.97E +0.60AC +0.29AD +0.64AE -0.96BC +0.66BD
-0.05BE -0.49CD -1.41CE -0.29DE -0.43ACD -0.17ACE +0.75ADE -1.64BCE +1.13BDE -0.58CDE +1.62ACDE Equation 3

Where A, B, C, D, and E denote temperature, time, S/S ratio, pH and % ethanol concentration, respectively.

Table 2 shows the analysis of variance (ANOVA), model summary, coefficient estimate and the effects of the selected factorial model for
screening of factors that significantly affect the antioxidant activity of the kalumpit extract. The F-value of 4.04 implies that the model is
significant and there is only a 0.63 % chance that an F-value this large could occur due to noise (p < 0.05). The "Lack of Fit F-value" of 0.84
implies the Lack of Fit is not significant relative to the pure error and that there is a 65.93 % chance that a "Lack of Fit F-value" this large
could occur due to noise (p > 0.05). The non-significant lack of fit indicates that the model fits and the “Adeq Precision” of 8.985 value, which
is greater than 4, is suggestive of an adequate signal. Therefore, the model generated can be used to navigate the design space.
A significant effect (p < 0.05) on the % DPPH radical scavenging activity of kalumpit extract was exhibited by temperature, S/S ratio, %
ethanol concentration, two-factorial interaction of S/S ratio and % ethanol concentration, three-factorial interactions of time, S/S ratio and %
ethanol concentration, and four-factorial interaction of temperature, S/S ratio, pH and % ethanol concentration. The effect of individual factors,
presented in Table 2, describes that the greater degree of effect is signified by a higher value. Temperature had the highest positive effect value
among all the factors and interactions with 13% contribution. The antioxidant activity of kalumpit extract increased when the temperature was
increased because heat enhances the diffusivity of extraction solvents into plant cells and stimulates higher solubility of phenolic compounds
in the extraction solvent (Chew et al., 2011). Similarly, the four-factorial interaction of temperature, S/S ratio, pH and % ethanol concentration
had significant positive effect on % DPPH scavenging activities of kalumpit extract with 9.19% contribution. In contrast, S/S ratio and %
ethanol had a high degree of negative effect with 16.19 and 13.55% contribution, respectively. The observed decrease in the antioxidant activity
of kalumpit extract, when the S/S ratio was increased, may be due to the limited amount of extractable phenolic compounds (Rezaei et al.,
2013). On the other hand, the observed negative effect of ethanol concentration is similar to the result reported by Li et al. (2019), wherein, an
increase in the ethanol concentration from 20% to 40% increased the antioxidant activity of Gordonia axillaris extract and gradually decreased
when the concentration was increased further to 80%. Increasing the volume of ethanol in water increases its polarity. Thus, in the case of
kalumpit extract, it is possible that most of the phenolic compounds present are moderately polar. Furthermore, in the extraction of bioactive
components, the use of a binary solvent system is found to be more favorable since water and alcohol can work in synergy. Water can swell
the plant matrix, thereby increasing the contact surface area between the solvent and the plant matrix (Alara et al., 2017).
The two-factorial interaction of S/S ratio and % ethanol concentration, three-factorial interaction time, S/S ratio and % ethanol concentration
also had significant negative effects with 6.9 and 9.47% contribution, respectively. With this, temperature, S/S ratio and % ethanol
concentration were the significant factors considered for the optimization of antioxidant extraction from kalumpit. Similarly, Belwal et al.
(2016) reported that the effects of extraction temperature, S/S ratio, and solvent concentration significantly affect the antioxidant activities of
Berberis asiatica fruits using ABTS, FRAP and DPPH Assays. Moreover, the same variables were optimized by Karacabey and Mazza
(2010) and Ilaiyaraja et al. (2015) on the extraction of phenolic compounds with high antioxidant activity from grape cane and wood apple,
respectively.

Response surface model of % DPPH radical scavenging activity of Kalumpit Extract

Optimization of the extraction process was carried out by applying the Box-Behnken design as shown in the experimental data in Table 3.
Table 4 shows the model for % DPPH radical scavenging activity with an R² of 0.9147 which indicates that 91.47% of the proportion of
variability in the response can be attributed to the model. Design Expert version 10.0.3 suggested a quadratic model represented by Equation
4. The Model F-value of 39.34 implies that the model was significant (p < 0.05) with a 0.01% chance that an F-value this large could occur
due to noise. The "Lack of Fit F-value" of 0.77 implies that the Lack of Fit was not significant (p > 0.05) relative to the pure error and there
was a 70.87% chance that a "Lack of Fit F-value" this large could occur due to noise. The model’s Adeq precision of 21.031 indicates an
adequate signal to noise ratio and that the model can be used to navigate the design space.

% DPPH Scavenging Activity= +62.69 +1.15A -3.70B -7.23C +0.35AB -0.35AC -5.32BC +0.13A2 -0.87B2 -6.89C2 Equation 4

Where A, B, and C denote temperature, S/S ratio and % ethanol concentration, respectively.

Table 1 2-level factorial experimental design for the screening of factors affecting the antioxidant activity of kalumpit extract
 Standard Run Temperature Time Solvent to Sample pH Ethanol DPPH Radical
Order Order (°C) (min) Ratio (mL∙g-1) Concentration Scavenging Activity
35 1 52.5 60 30 5.5 60
(%) 74.07
(%)

28 2 80 90 10 8 100 73.88

21 3 25 30 50 3 100 65.5

10 4 80 30 10 8 20 71.64

27 5 25 90 10 8 100 70.18

32 6 80 90 50 8 100 66.18

19 7 25 90 10 3 100 64.81

18 8 80 30 10 3 100 65.79

8 9 80 90 50 3 20 72.51

15 10 25 90 50 8 20 68.03

2 11 80 30 10 3 20 68.13

1 12 25 30 10 3 20 67.64

3 13 25 90 10 3 20 71.05

20 14 80 90 10 3 100 72.03

22 15 80 30 50 3 100 67.15

13 16 25 30 50 8 20 67.15

9 17 25 30 10 8 20 67.06

14 18 80 30 50 8 20 65.5

11 19 25 90 10 8 20 65.6

16 20 80 90 50 8 20 68.03

33 21 52.5 60 30 5.5 60 67.74

12 22 80 90 10 8 20 69.59

23 23 25 90 50 3 100 56.14

26 24 80 30 10 8 100 66.08

29 25 25 30 50 8 100 55.26

17 26 25 30 10 3 100 63.74

7 27 25 90 50 3 20 63.65

34 28 52.5 60 30 5.5 60 69.4

5 29 25 30 50 3 20 60.43

6 30 80 30 50 3 20 69.98

30 31 80 30 50 8 100 61.7

24 32 80 90 50 3 100 57.7

4 33 80 90 10 3 20 66.37

25 34 25 30 10 8 100 61.7

31 35 25 90 50 8 100 51.56

Table 2 Analysis of variance, model summary, and estimated effects of selected factorial model affecting antioxidant activity of kalumpit
extract
Source/Factor df
 Sum of Mean F p-value Coefficient Standardized %
Squares Square Value Prob > F Estimate Effect Contribution

Model 792.59 21 37.74 4.04 0.0063

A-Temperature 123.12 1 123.12 13.18 0.0030 1.96 3.92 13.47

B-Time 5.17 1 5.17 0.55 0.4700 0.4 0.80 0.57

C- Solvent to Sample Ratio 147.97 1 147.97 15.84 0.0016 -2.15 -4.30 16.19

D-pH 0.38 1 0.38 0.041 0.8423 -0.11 -0.22 0.042

E- Ethanol Conc. 123.89 1 123.89 13.26 0.0030 -1.97 -3.94 13.55

AC 11.64 1 11.64 1.25 0.2845 0.6 1.21 1.27

AD 2.74 1 2.74 0.29 0.5975 0.29 0.59 0.3

AE 13.09 1 13.09 1.4 0.2577 0.64 1.28 1.43

BC 29.27 1 29.27 3.13 0.1001 -0.96 -1.91 3.2

BD 13.85 1 13.85 1.48 0.2450 0.66 1.32 1.52

BE 0.096 1 0.096 0.01 0.9207 -0.055 -0.11 0.011

CD 7.79 1 7.79 0.83 0.3777 -0.49 -0.99 0.85

CE 63.91 1 63.91 6.84 0.0214 -1.41 -2.83 6.99

DE 2.62 1 2.62 0.28 0.6051 -0.29 -0.57 0.29

ACD 5.99 1 5.99 0.64 0.4378 -0.43 -0.87 0.65

ACE 0.93 1 0.93 0.1 0.7572 -0.17 -0.34 0.1

ADE 17.96 1 17.96 1.92 0.1888 0.75 1.50 1.97

BCE 86.57 1 86.57 9.27 0.0094 -1.64 -3.29 9.47

BDE 40.64 1 40.64 4.35 0.0573 1.13 2.25 4.45

CDE 10.94 1 10.94 1.17 0.2988 -0.58 -1.17 1.2

ACDE 84.02 1 84.02 8.99 0.0103 1.62 3.24 9.19

Residual 121.43 13 9.34

Lack of Fit 99.84 11 9.08 0.84 0.6593

Pure Error 21.59 2 10.79

Cor Total 914.02 34

Model Summary

Std. Dev. 3.06 R-Squared 0.8671 Adeq Precision 8.985

Mean 66.08 Adj R-Squared 0.6525 BIC 221.08

C.V. % 4.62 Pred R-Squared 0.4266 AICc 271.2

PRESS 524.13 -2 Log Likelihood 142.87

Table 4 shows that the linear terms of temperature, S/S ratio, ethanol concentration, interactive effect of S/S ratio, ethanol concentration, and
quadratic effect of ethanol concentration were found to significantly affect the % DPPH radical scavenging activity of the kalumpit extract (p
< 0.05). The significant linear effects of extraction temperature and ethanol concentration on the % DPPH radical scavenging activity are
consistent with the reports of Belwal et al. (2016), Ilaiyaraja et al. (2015), Karacabey and Mazza (2010), and Li et al. (2012) on the
antioxidant extraction from Berberis asiatica fruits, wood apple, grape, and tomato, respectively. On the other hand, the quadratic effect of
ethanol concentration agrees with the report of Belwal et al. (2016) on the significant quadratic effect of solvent on FRAP activity of Berberis
asiatica fruit extract.

Positive values of coefficients indicate a positive effect on the response variable while negative coefficients indicate the opposite. Higher
coefficient values indicate greater contribution to the response. In this regard, extraction temperature had a significant positive effect on the %
DPPH radical scavenging activity of kalumpit extract (p < 0.05), demonstrated by its coefficient estimate of 1.15 (Table 4) and as shown in
Figure 1A. It can be observed that high level of temperature (80 °C) resulted in high % DPPH scavenging activity. This can be attributed to
the higher diffusion coefficient of phenolic compounds, resulting in high solubility and extraction rate (Ju and Howard, 2003; Pompeu et al.,
2009; Piovesan et al., 2017). This result agrees with several studies that reported on the positive effect of increasing extraction temperature on
antioxidant activity (Vatai et al. 2009; Li et al., 2012; Benmeziane et al. 2013; Piovesan et al., 2017).
On the other hand, linear effect of S/S ratio was found to have significant negative effect (p < 0.05) on % DPPH scavenging activity of kalumpit
extract as shown in Table 4 and Figure 1B. It can be observed that low level S/S ratio (10 mL/g) resulted in high % DPPH scavenging activity.
The significant negative effect of S/S ratio in this study is consistent with the findings of Kemerli-Kalbaran and Ozdemir (2019) on the
optimization of DPPH scavenging activities of lipophilic and hydrophilic phase, and oil of pine nut.
The linear and quadratic effects of ethanol concentration were observed to have significant negative effects on % DPPH scavenging activity
of kalumpit extract as shown in Table 4 and Figure 1C. In this regard, ethanol concentration near the midpoint level resulted in high % DPPH
scavenging activity. Among interactive effects, only the interaction of S/S ratio and ethanol concentration was found to have significant
negative effects as shown in Table 4. Figure 2C shows that high % DPPH scavenging activity can be observed in low-level S/S ratio and
midpoint-level of ethanol concentration. The significant negative effect of ethanol concentration, in quadratic term, is consistent with the
report of Lee et al. (2016) on % DPPH scavenging activity of Korean red ginseng. Increasing ethanol concentration up to the midpoint level
results in reduced dielectric constant of the solution and energy needed to separate solvent molecules, allowing solutes to easily dissolve in the
solvent (Cacace and Mazza, 2002, 2003; Pompeu et al., 2009)
The interaction of temperature and S/S ratio had a positive effect while the interaction of S/S ratio and ethanol concentration showed negative
effect as shown in Table 4. However, both effects were not significant. Figure 2A shows that high % DPPH scavenging activity can be observed
in high-level extraction temperature and low-level S/S ratio. Moreover, Figure 2B shows that a high % DPPH scavenging activity can be
observed in high-level extraction temperature and midpoint-level of ethanol concentration.

Table 3 Box-Behnken design for optimization of DPPH and ABTS scavenging activities of kalumpit extract

Solvent to Sample Ethanol DPPH Radical ABTS Scavenging

Standard Run Temperature
Block Ratio Concentration Scavenging Activity Activity
Order Order (°C)
(mL∙g-1) (%) (%) (%)

7 1 1 25 30 100 53.08 2.50

1 1 2 25 10 60 62.31 66.43

6 1 3 80 30 20 65.38 52.86

9 1 4 52.5 10 20 59.62 79.64

13 1 5 52.5 30 60 63.08 76.43

10 1 6 52.5 50 20 62.31 21.79

11 1 7 52.5 10 100 61.54 26.43

5 1 8 25 30 20 62.69 5.36

2 1 9 80 10 60 65.38 78.93

12 1 10 52.5 50 100 38.08 5.71

14 1 11 52.5 30 60 55.00 56.79

15 1 12 52.5 30 60 62.31 34.64

8 1 13 80 30 100 46.54 13.57

4 1 14 80 50 60 60.77 41.43

3 1 15 25 50 60 57.69 2.50

31 2 16 25 10 60 63.46 77.14

44 2 17 52.5 30 60 63.46 76.79

43 2 18 52.5 30 60 62.31 50.71

33 2 19 25 50 60 58.08 8.93

38 2 20 80 30 100 49.23 31.43

41 2 21 52.5 10 100 56.92 30.36

40 2 22 52.5 50 20 60.77 17.14

42 2 23 52.5 50 100 39.23 4.29

35 2 24 25 30 20 62.69 19.64

36 2 25 80 30 20 63.85 46.43
 45 2 26 52.5 30 60 63.08 41.79

34 2 27 80 50 60 59.62 23.57

39 2 28 52.5 10 20 61.15 79.64

32 2 29 80 10 60 65.77 79.29

37 2 30 25 30 100 43.85 3.57

21 3 31 80 30 20 67.69 34.64

26 3 32 52.5 10 100 58.46 11.43

18 3 33 25 50 60 57.69 18.93

19 3 34 80 50 60 62.69 30.71

28 3 35 52.5 30 60 67.69 70.71

30 3 36 52.5 30 60 64.23 63.57

29 3 37 52.5 30 60 63.08 37.86

25 3 38 52.5 50 20 61.54 15.36

16 3 39 25 10 60 65.00 51.79

17 3 40 80 10 60 65.00 75.36

23 3 41 80 30 100 49.23 27.14

24 3 42 52.5 10 20 61.15 82.14

20 3 43 25 30 20 63.08 4.29

27 3 44 52.5 50 100 38.46 12.14

22 3 45 25 30 100 43.85 5.00

Figure 1 Model graphs for linear effects of extraction temperature (A), S/S ratio (B) and ethanol concentration (C) on % DPPH scavenging
activity of kalumpit extract

Table 4 Analysis of variance and model summary for response surface quadratic model for % DPPH scavenging activity of kalumpit extract
 Source Sum of df Mean F Value p-value Coefficient
Squares Square Prob > F Estimate
Block 9.18 2 4.59

Model 2489.7 9 276.63 39.34 < 0.0001

A-Temperature 31.95 1 31.95 4.54 0.0406 1.15

B-Solvent to Sample Ratio 328.9 1 328.9 46.78 < 0.0001 -3.7

C-Ethanol Concentration 1253.7 1 1253.7 178.3 < 0.0001 -7.23

AB 1.49 1 1.49 0.21 0.6481 0.35

AC 1.49 1 1.49 0.21 0.6481 -0.35

BC 339.69 1 339.69 48.31 < 0.0001 -5.32

A2 0.18 1 0.18 0.026 0.8731 0.13

B2 8.3 1 8.3 1.18 0.2853 -0.87

C2 526 1 526 74.81 < 0.0001 -6.89

Residual 232.03 33 7.03

Lack of Fit 180.06 27 6.67 0.77 0.7087

Pure Error 51.97 6 8.66

Cor Total 2730.91 44

Model Summary

Std. Dev. 2.65 R-Squared 0.9147

Mean 58.62 Adj R-Squared 0.8915

C.V. % 4.52 Pred R-Squared 0.8461

PRESS 418.88 Adeq Precision 21.031

-2 Log Likelihood 201.51 BIC 247.19

AICc 235.26
Figure 2 Contour plots for interaction effects of extraction temperature and solvent to sample ratio (A), temperature and ethanol concentration
(B), and solvent to sample ratio and ethanol concentration (C) on % DPPH scavenging activity of kalumpit extract

Response surface model of % ABTS radical scavenging activity of Kalumpit Extract

Table 5 shows the ANOVA table for the special quadratic model for % ABTS radical scavenging activity with an R² of 0.8637. The Model F-
value of 23.23 implies that the model is significant (p < 0.05) and there is only a 0.01% chance that an F-value this large could occur due to
noise. The "Lack of Fit F-value" of 0.24 implies that the Lack of Fit is not significant (p > 0.05) relative to the pure error and there is a 99.54%
chance that a "Lack of Fit F-value" this large could occur due to noise. Moreover, the model’s “Adeq precision” of 13.395 indicates an adequate
signal to noise ratio and that the model can be used to navigate the design space.
Equation 5 and Table 5 describe the special quadratic model for % ABTS scavenging activity of kalumpit extract. The linear effects of
temperature, S/S ratio, ethanol concentration, interactive effect of S/S ratio and ethanol concentration, and quadratic effects of extraction
temperature and ethanol concentration were found to significantly influence the % ABTS scavenging activity of kalumpit extract (p < 0.05).
Moreover, the linear effect of extraction temperature and interactive effect of S/S ratio and ethanol concentration on % ABTS scavenging
activity of kalumpit extract were observed to be significantly positive as evidenced by their coefficient estimate values of 11.22 and 11.76,
respectively. Figure 3A shows that a high % ABTS scavenging activity of kalumpit extract can be observed in midpoint level of extraction
temperature. The positive effect of temperature can be attributed to the reduced viscosity and surface tension of the solvent, resulting in higher
phenolic solubility, extraction and diffusion rate, and antioxidant activity (Ju and Howard, 2003; Piovesan et al., 2017). This was also found
to be consistent with Lee et al. (2016) report on the significant positive effect of extraction temperature on the % DPPH scavenging activity of
Korean red ginseng extract. Figure 4C show that a high % ABTS scavenging activity of kalumpit extract can be observed in low-level S/S ratio
and midpoint-level of ethanol concentration. Although insignificant, the interaction between temperature and S/S ratio also have positive effects
as shown in Figure 4A and confirmed by a coefficient estimate of 2.26 (Table 5). A medium level % ABTS scavenging activity of kalumpit
extract can be observed at midpoint-level of extraction temperature and low-level S/S ratio.
On the other hand, the linear terms of S/S ratio and ethanol concentration, and the quadratic terms of extraction temperature and ethanol
concentration, were found to have significant negative effects on the % ABTS scavenging activity of kalumpit extract. In this regard, a high %
ABTS scavenging activity of kalumpit extract can be observed at midpoint level of extraction temperature (Figure 3A), low-level S/S ratio
(Figure 3B) and midpoint-level of ethanol concentration (Figure 3C). The linear significant negative effects of S/S ratio and ethanol
concentration agrees with Cheok et al. (2012) report on the negative effects of S/S ratio and methanol concentration on total phenolic content
of Garcinia mangostana Linn. hull. On the other hand, the significant quadratic effect of the ethanol concentration on % ABTS scavenging
activity of kalumpit extract is consistent with Lee et al. (2016) and Liyana-Pathirana and Shahidi (2005) report on the significant negative
effect of ethanol concentration quadratic term on antioxidant activity of Korean red ginseng and hard wheat, respectively.
Interaction between extraction temperature and ethanol concentration also had an insignificant negative effect on the % ABTS scavenging
activity of kalumpit extract, as demonstrated by its coefficient estimate of -3.63. An intermediate % ABTS scavenging activity of kalumpit
extract can be observed at midpoint level of extraction temperature and ethanol concentration as shown in Figure 4B.

% ABTS Scavenging Activity= +56.59 +11.22A -22.34B -11.89C +2.26AB -3.63AC +11.76C -10.99A2 +0.65B2 -25.06C2 Equation 5

Where A, B, and C denote temperature, S/S ratio and % ethanol concentration, respectively.

Table 5 Analysis of variance and model summary for response surface quadratic model for % ABTS scavenging activity of kalumpit extract

Sum of Mean p-value

Source df F Value Coefficient
Squares Square Prob > F
Estimate

Block 82.18 2 41.09

Model 28255.97 9 3139.55 23.23 < 0.0001

A-Temperature 3021.45 1 3021.45 22.36 < 0.0001 11.22

B- Solvent to sample ratio 11973.86 1 11973.86 88.61 < 0.0001 -22.34

C-Ethanol Concentration 3392.86 1 3392.86 25.11 < 0.0001 -11.89

AB 61.39 1 61.39 0.45 0.505 2.26

AC 158.21 1 158.21 1.17 0.2871 -3.63

BC 1658.43 1 1658.43 12.27 0.0013 11.76

A2 1337.17 1 1337.17 9.9 0.0035 -10.99

B2 4.68 1 4.68 3.50E-02 0.8535 0.65

C2 6958.84 1 6958.84 51.5 < 0.0001 -25.06

Residual 4459.16 33 135.13

Lack of Fit 2326.34 27 86.16 0.24 0.9954

Pure Error 2132.82 6 355.47

Cor Total 32797.32 44

Model Summary

Std. Dev. 11.62 R-Squared 0.8637

Mean 37.71 Adj R-Squared 0.8265

C.V. % 30.83 Pred R-Squared 0.7663

PRESS 7646.64 Adeq Precision 13.395

-2 Log Likelihood 334.53 BIC 380.21

AICc 368.28
Figure 3 Model graphs for linear effects of extraction temperature (A), solvent to sample ratio (B) and ethanol concentration (C) on % ABTS
scavenging activity of kalumpit extract

Figure 4 Contour plots for interaction effects of extraction temperature and solvent to sample ratio (A), temperature and ethanol concentration
(B), and solvent to sample ratio and ethanol concentration (C) on % ABTS scavenging activity of kalumpit extract
Optimization and validation of extraction models developed

Ultimately, the models for % DPPH and ABTS radical scavenging activities of kalumpit fruit extract were confirmed to adequately fit the
response surface, hence, no evidence supports the claim that the model does not adequately explain the variation in the response. The composite
desirability of 0.977 is indicative that the settings for the factors achieved favorable results for all responses as a whole and the goal for
optimization was satisfied. A desirability function approach as a numerical optimization technique was used to determine the optimum
extraction condition where the highest % DPPH and ABTS radical scavenging activities of kalumpit fruit extract were attained. Constraints
were defined as the lower and upper limit range of extraction temperature, S/S ratio and ethanol concentration to attain maximum % DPPH
and ABTS radical scavenging activities and desirability. Based on the numerical optimization with the desirability function and the constraints
given above, the optimum condition of extraction was attained at the extraction temperature of 80.0 °C, S/S ratio of 10 mL g -1 min and ethanol
concentration of 51.66%, which resulted in the maximum desirability value of 0.977, and predicted % DPPH and ABTS radical scavenging
activities of 66.63 and 82.14, respectively.
The adequacy of the predictive models at the optimum condition was validated by performing 3 independent experiments while the
experimental data were compared with the predicted values (Table 6). No significant difference was found between experimental data and
predicted values (p > 0.05) indicating that the models developed were adequate to describe the relationship between the factors and responses.
Moreover, less than 5% error was observed for both response parameters.

Table 6 T-test analysis for validation of the optimized conditions for extraction of antioxidants from kalumpit.
Response Parameter Predicted Experimental t- Value p-Value % Error

% DPPH scavenging activity 66.63±2.65 67.66±2.05 -0.53 0.632 1.54

% ABTS scavenging activity 82.10±11.60 78.98±6.57 0.41 0.709 -3.80

CONCLUSION

The results showed that temperature, S/S ratio and % ethanol are significant factors that affect the extraction of antioxidants from kalumpit. In
addition, optimum % DPPH and ABTS radical scavenging activities of kalumpit extract were generally obtained at high-level extraction
temperature, low-level S/S ratio and midpoint-level ethanol concentration. Predictive models adequately described the % DPPH and ABTS
radical scavenging activities of kalumpit extract as a function of independent factors. The desirability function approach determined the
optimum conditions for the extraction of kalumpit resulting in maximum % DPPH and ABTS radical scavenging activities. The optimum
conditions for the solvent extraction of antioxidants were attained at the extraction temperature of 80.0 °C, S/S ratio of 10 mL g-1 min and
ethanol concentration of 51.66% resulting in the maximum desirability value of 0.977, and predicted % DPPH and ABTS radical scavenging
activities of 66.63 and 82.14, respectively. The baseline data generated by this research is useful in maximizing the potential of kalumpit,
through the production of high-value food ingredients from kalumpit such as health promoting antioxidants and natural colorants.

Acknowledgment: Authors are grateful to UP OVPAA-ECWRG (University of the Philippines Office of the Vice President for Academic
Affairs- Enhanced Creative Work and Research Grant) for providing economical support.

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