Preparative Biochemistry and Biotechnology

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Optimization of the extraction of polyphenols and

antioxidant activity from Malva parviflora L. leaves
using Box–Behnken design

Ekram A. Abd El-Salam & Nashwa F. S. Morsy

To cite this article: Ekram A. Abd El-Salam & Nashwa F. S. Morsy (2019): Optimization
of the extraction of polyphenols and antioxidant activity from Malva�parviflora L.
leaves using Box–Behnken design, Preparative Biochemistry and Biotechnology, DOI:
10.1080/10826068.2019.1633667

To link to this article: https://doi.org/10.1080/10826068.2019.1633667

Published online: 27 Jun 2019.

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PREPARATIVE BIOCHEMISTRY AND BIOTECHNOLOGY
https://doi.org/10.1080/10826068.2019.1633667

Optimization of the extraction of polyphenols and antioxidant activity from

Malva parviflora L. leaves using Box–Behnken design
Ekram A. Abd El-Salam and Nashwa F. S. Morsy
Faculty of Agriculture, Food Science Department, Cairo University, Giza, Egypt

ABSTRACT KEYWORDS
Box–Behnken Design (BBD) was used to optimize the extraction conditions of polyphenols from Antioxidants; Box–Behnken
Malva (Malva parviflora L.) leaves. The effect of ethanol concentration (20–80%), solvent/leaf pow- design; Malva parviflora L.;
der ratio (10:1 to 30:1, v/w) and extraction time (5–45 min) on the polyphenols yield and the 2,2- polyphenols
diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of the obtained extracts were investi-
gated. Quadratic models fit well. The optimal conditions (53.40% ethanol, solvent/leaf powder
ratio 20:1 (v/w), and 15 min) resulted in an extract with a maximum yield of polyphenols
(1098.4 mg GAE/100 g leaf powder) and high inhibition percentage of DPPH radical (33.31%) with
desirability 0.742. High Performance Liquid Chromatography (HPLC) analysis indicated that the
major identified polyphenol compounds extracted at the optimal conditions were naringenin,
q-coumaric acid, apigenin-7-glucoside, luteolin, and cinnamic acid. These findings indicate that M.
parviflora leaf extracts possess DPPH radical scavenging activity and could be used as a natural
source for bioactive products.

Introduction polyphenols using various extraction techniques from differ-

ent materials.[10,14–16] Box–Behnken Design (BBD) is a type
Numerous investigations focused on antioxidants from natural
of RSM that reduces the experiments required to find the
sources. Recently, attention has been paid to polyphenols as
optimal conditions and illustrates the interactions among
biologically active ingredients, due to its antioxidant, anti-
process variables. BBD is applied as an exploratory method
inflammatory and antitumor activities.[1] Polyphenols include
for second-order experiments to establish a quadratic
a lot of compounds that originated in the plant kingdom and
response surface.[17–19]
differ in their structures and functions.[2] Several studies were
Based on our knowledge, there is no available research
conducted to investigate the content and beneficial effects of
concerning the extraction optimization of Malva parviflora
polyphenols in different materials.[2–4] Extraction is the first
L. leaf for the efficient recovery of bioactive compounds,
step in the recovery of polyphenols. The optimum conditions
such as polyphenols, using aqueous ethanol. Preliminary sin-
for extracting polyphenols differ according to raw material
gle factor experiments were used to find suitable ranges of
and physicochemical factors.[5–11]
the extraction variables (extraction time, solvent/leaf powder
Malva parviflora L. (Malvaceae family) is a wild herb that
ratio, and ethanol concentration). BBD was used to establish
is prevalent in North Africa. It is known as little mallow
combinations of the selected ranges of the independent
and cheeseweed mallow.[12] Its common name in Egypt is
extraction factors to obtain polyphenols rich extract with
Khoubbiza. Malva leaves are traditionally cooked like spin-
high DPPH radical scavenging activity from Malva leaf.
ach and also used in the treatment of the flu, fever, dysen-
Furthermore, polyphenols extracted with the optimal condi-
tery, and wounds.[13]
tions were identified and quantified by High Performance
Conventional extraction methods of polyphenols with
Liquid Chromatography.
one factor at a time have disadvantages such as lengthy dur-
ation, low extraction efficiency, and decomposition of
thermolabile compounds. These methods neglect the inter- Materials and methods
action effect among the variables and lead to misinterpret-
Plant material
ation of results. The response surface methodology (RSM) is
an effective statistical technique for optimizing the responses Malva parviflora L. leaves were collected in October 2017,
that are affected by the independent variables. It can predict from Geziret El-Dahab, Giza, Egypt; Latitude 29
590
interaction effects of variables on process response. It is suc- 0.952800 (N), Longitude 31
130 19.613400 (E). The leaves were
cessfully used to optimize extraction conditions of manually separated and washed before drying at 40
C in an

CONTACT Ekram A. Abd El-Salam eaam2000@agr.cu.edu.eg Food Science Department, Faculty of Agriculture, Cairo University, Giza 12613, Egypt.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lpbb.
ß 2019 Taylor & Francis Group, LLC
2 E. A. ABD EL-SALAM AND N. F. S. MORSY
oven (Heraeus electronics, Germany) for 20 hr. The dried
leaves were grounded in a bench top analytical mill (Cole-
Parmer, USA), sieved through 0.500 mm stainless steel sieve
and stored at 8
C until analysis.
Chemicals
Gallic acid, Folin-Ciocalteu reagent, and 2,2-diphenyl-1-pic-
rylhydrazyl (DPPH) and HPLC-grade acetonitrile were pur-
chased from Sigma Chemical Co., Ltd (St. Louis, MO, USA).
All other reagents and solvents were of analytical grade.
Preliminary single factor extraction experiments
The Malva leaf powder was extracted at ambient tempera-
ture using magnetic stirring (Medline Scientific, Model MS
300, UK). The leaf powder (3.33–10 g) was mixed with
100 mL of ethanol solution (20–80%) corresponding to solv-
ent/leaf powder ratio of 10:1–30:1 (v/w), for time ranged
from 5 to 45 min with 5 min intervals. The extract was fil-
tered through Whatman filter paper No. 1 before analysis.
Figure 1. Preliminary experiments of each extraction parameter level’s effect on total polyphenols content [TPC (mg GAE/100 g dried leaf powder)] in Malva leaf
powder: (A) extraction time (min), (B) solvent/leaf powder ratio (mL/g), and (C) ethanol concentration (%).
All single factor experiments were performed in triplicate.
One-way ANOVA followed by Tukey’s test at p < 0.05 level
was used to estimate the significant effect of different factor
levels (Minitab 17, Minitab Inc., USA). Three factor levels
with the highest extracted polyphenols were used for further
RSM design.
Box–Behnken design
Based on the results of the preliminary experiments (as
shown in Figure 1) the levels of the independent variables
that were selected are depicted in Table 1: time (X1,
5–15 min), solvent/leaf powder ratio (X2, 20:1–30:1), and
solvent concentration (X3, 40–60%).
BBD with three factors (X1, X2, X3) at three levels (–1, 0,
þ1) was used to optimize the extraction process of total pol-
yphenols and its corresponding scavenging activity against
DPPH radicals. The experimental design consists of 17 com-
binations (Table 1) including five replicates of the central
point to estimate the pure error. All the experiments were
conducted in triplicate.
 PREPARATIVE BIOCHEMISTRY AND BIOTECHNOLOGY 3

Table 1. The Box–Behnken design with experimental data for the extraction of bioactive compounds from Malva.
DPPH radical inhibition percentage of
Time Solvent ratio X Solvent conc. Yield of total polyphenols (mg Concentration of polyphenols 1 mL extract
Run (min) (mL): 1 solid (Ethanol %) GAE/ 100 g dried leaf powder) (mg GAE/ mL extract) the extract containing 1 mg GAE
1 5 (–1) 20 (–1) 50 (0) 786i ± 14 393.00gh ± 7 29.85b ± 0.12 76.0a ± 1.1
2 15 (þ1) 20 (–1) 50 (0) 1006ef ± 22.4 503.03a ± 11.21 35.28a ± 1.19 70.1bc ± 2.1
3 5 (–1) 30 (þ1) 50 (0) 1180a ± 12.12 393.33gh ± 4.04 22.63d ± 0.28 57.5d ± 1.2
4 15 (þ1) 30 (þ1) 50 (0) 1122ab ± 18.7 374.00hi ±6.24 17.66h ± 0.17 47.2g ± 0.8
5 5 (–1) 25 (0) 40 (–1) 1091bcd ± 31.3 436.50def ±12.5 20.59efg ± 0.18 47.2g ± 1.1
6 15 (þ1) 25 (0) 40 (–1) 1035de ± 25 414.00fg ±10 19.59g ± 0.45 47.4g ± 2.0
7 5 (–1) 25 (0) 60 (þ1) 892gh ± 15.61 357.00i ± 6.24 24.28c ± 0.04 68.0c ± 1.1
8 15 (þ1) 25 (0) 60 (þ1) 1046cde ± 21.3 418.67efg ± 8.5 20.18fg ± 0.48 48.2fg ± 0.8
9 10 (0) 20 (–1) 40 (–1) 949fg ± 25 474.50bc ± 12.5 20.62dfg ± 0.11 43.5h ± 1.0
10 10 (0) 30 (þ1) 40 (–1) 1074bcd ± 15 358.00i ± 5.00 17.75h ± 0.11 49.6efg ± 0.6
11 10 (0) 20 (–1) 60 (þ1) 975f ± 15 487.50ab ± 7.50 24.89c ± 0.25 51.1efg ± 0.9
12 10 (0) 30 (þ1) 60 (þ1) 888h ± 15.59 296.00j ± 5.20 21.34e ± 0.11 72.1b ± 1.3
13 10 (0) 25 (0) 50 (0) 1086bcd ± 14.22 413.00fg ± 8.00 21.53de ± 0.24 52.7e ± 0.9
14 10 (0) 25 (0) 50 (0) 1098bc ± 26.3 439.50def ± 10.5 21.20ef ± 0.07 48.3fg ± 1.1
15 10 (0) 25 (0) 50 (0) 1090bcd ± 12.5 448.00cd ± 17 21.73de ± 0.26 48.5fg ± 1.3
16 10 (0) 25 (0) 50 (0) 1089bcd ± 9.46 420.50defg ± 10.5 21.05ef ± 0.30 50.4efg ± 1.2
17 10 (0) 25 (0) 50 (0) 1104bc ± 16.65 442.50de ± 6.5 20.96ef ± 0.28 47.7fg ± 0.8
P (Brown-Forsythe) 0.993 0.960 0.609 0.999
P (ANOVA) < 0.001 < 0.001 < 0.001 <0.001
Means followed by different letters in the same column significantly differ based on Tucky’s test at p < 0.05.

Brown-Forsythe test and one-way ANOVA followed by Radical scavenging activity

Tukey’s test at p < 0.05 level were used to evaluate the
homogeneity of variance for all responses and the significant
effect of different combinations, respectively (Minitab 17,
Minitab Inc., USA). To implement BBD, Design-Expert ver-
sion 7.0.0 (Stat-Ease, Inc., Minneapolis, MN, USA) was
used.[20] The experimental data were fitted using the quad-
ratic model [Eq. (1)], that has the following equation:
X
3 X
3 XX
3 ¼
Y ¼ b0 þ bj Xj þ bjj Xj2 þ bij Xi Xj þ ei
j¼1 j¼1 i <j ¼ 2
where, Y is the dependent variable. Xi and Xj are coded
independent variables. b0, bj, bjj, and bij are variable regres-
sion coefficients for the model intercept, linear, quadratic,
and interaction effects, respectively. The error is represented
by ei.[21] The adequacy of the generated mathematical
models was evaluated regarding their determination
coefficients R2; R2adj ; predicted R2, (CV%), and adequate
precision values.
Total polyphenols content
The total polyphenols content (TPC) of the Malva leaf
extract was determined by the Folin-Ciocalteu method at
room temperature using Gallic acid as a standard.[22]
Properly diluted sample (10 lL) was mixed using vortex
with 50 lL of Folin-Ciocalteu reagent and 790 lL of distilled
water. After 1 min, 150 lL of 20% sodium carbonate solution
were added, vortexed and left to stand in a dark place for
120 min. Absorption was read at 750 nm (UV-2000
Spectrophotometer, Unico, USA). Calibration curve (r2 ¼
0.9990) of standard Gallic acid (50–800 mg/L) was used to
calculate the TPC. Results were expressed as mg Gallic acid
equivalents (GAE)/100 g dry leaf powder and mg Gallic acid
equivalents (GAE)/mL extract.
The scavenging activity of properly diluted Malva leaf pow-
der extracts towards DPPH radicals was assessed spectro-
photometrically (UV-2000 Spectrophotometer, Unico, USA)
at 515 nm against a blank.[23] The inhibition percentage of
DPPH radical was calculated using Eq. (2).
Inhibition percentage of DPPH radical
 
A517nm of control A517 nm of sampl (2)
 100
A517 nm of control
(1)
The correlation between inhibition percentage of DPPH
radical and its corresponding polyphenols at extract concen-
tration 1 mg GAE/mL extract was calculated using Eq. (3)
Inhibition percentage of DPPH radical of 1mg GAE=mL extract
Inhibition percentage of DPPH radical
¼  
Total polyphenols of the extract mg GAE=mL extract
(3)
HPLC analysis of polyphenols
Polyphenols of Malva leaf extract obtained at the optimum
conditions were quantified.[24] HPLC-DAD (Agilent
Technologies 1100 series liquid chromatograph, USA)
equipped with an auto sampler was used. The Eclipse XDB-
C18 column (150  4.6 mm; 5 mm) was used with a C18 guard
column (Phenomenex, Torrance, CA). The mobile phase
consisted of acetonitrile (solvent A) and 2% acetic acid in
water (v/v) (solvent B). Elution was performed at a flow rate
of 0.8 mL/min. The gradient program consisted of 0–100%
A in B over 0–60 min. The injection volume was 50 mL, and
the peaks were monitored simultaneously at 280 and 320 nm
for the benzoic acid and cinnamic acid derivatives, respect-
ively as well as 360 nm for flavonoids. All samples were fil-
tered through a 0.45 mm Acrodisc syringe filter (Gelman
Laboratory, MI) before injection. The peaks were identified
4 E. A. ABD EL-SALAM AND N. F. S. MORSY
by retention time after matching with the standards. The
linear calibration curve of the standards was used for
quantification.
Results and discussion
Preliminary single factor experiments
Extraction time
The effect of extraction time (5–45 min) on the yield of poly-
phenols from the Malva leaf powder was investigated using
ethanol concentration 80% (v/v); and solvent/leaf powder ratio
20:1 (v/w). Results are illustrated in Figure 1A. Extending
extraction time from 5 to 10 min improved significantly (p ˂
0.05) the extraction efficiency of polyphenols, after which no
further increase was obtained. This could be due to the small
particle size leaf powder that enhanced mass transfer process
and decreased the time for maximum extraction.[25] The max-
imum yield of polyphenols was found to be 581.33 ± 10.26 mg
GAE/100 g leaf powder. Based on the obtained results, 5, 10,
and 15 min were selected for RSM experiments.
Solvent/leaf powder ratio (v/w)
Polyphenol yields with different solvent/leaf powder ratios
(10:1 to 30:1, v/w) were evaluated at extraction time 10 min
and ethanol concentration 80% (v/v). As shown in Figure
1B, increasing solvent/material ratio from 10:1 to 20:1 (v/w)
increased significantly (p ˂ 0.05) the yield of polyphenols
from 423.3 ± 21.5 to 556.67 ± 9.02 mg GAE/100 g leaf pow-
der. Mass transfer of the extractable material is highly
dependent on its concentration in the solvent until it
reached equilibrium.[26,27] Results revealed that the continual
increase of solvent/material ratio to 30:1 (v/w) did not
enhance significantly (p ˂ 0.05) the extraction of polyphe-
nols. When the mass transfer of the extractable material is
maximized, any further increase of solvent/material ratio
(v/w) does not enhance the extraction yield.[28,29] Thus, the
solvent/leaf powder ratio in the RSM experiments was deter-
mined at 20:1, 25:1, and 30:1 (v/w).
Effect of solvent concentration
The effect of different concentrations of aqueous ethanol
(from 20 to 80%) was evaluated using 10 min extraction
time and solvent/leaf powder ratio of 20:1 (v/w). As shown
in Figure 1C, increasing the ethanol concentration to 40%
increased significantly (p ˂ 0.05) the yield of polyphenols to
the maximum level (990.66 ± 13.29 mg GAE/100 g leaf pow-
der). Extracts obtained with solvent concentrations of 40%
and 50% had significantly (p ˂ 0.05) the same level of poly-
phenols. Increasing the ethanol concentration to levels
higher than 50% exhibited a significant (p ˂ 0.05) decrease
in the extraction efficiency of polyphenols. The extraction of
polyphenols is improved using binary solvent systems (etha-
nol/water) instead of mono-solvent systems due to their
relative polarity.[30,31] Therefore, ethanol concentration of
40, 50, and 60% were used for RSM design.
Response surface methodology analysis
Average values of polyphenols and inhibition percentage of
DPPH radical are listed in Table 1. Brown-Forsythe’s test
indicated that all responses were homoscedastic (p  0.6),
while significant differences (p ˂ 0.001) were found for the
dependent variables using one-factor ANOVA.
The yield of polyphenols varied significantly (p < 0.001)
from 786 ± 14.00 to 1180 ± 12.12 (mg GAE/100 g dry leaf pow-
der). This result is higher than that obtained by Afolayan
et al.[32] who found that TPC was 290 mg GAE/100 g dried M.
parviflora whole plant. This difference may be explained by
part of the plant being extracted, type of solvent and the
extraction technique used. They used shaking technique dur-
ing extraction of whole plant with methanol for 24 hr.
Results in Table 1 indicate that the inhibition percentage of
DPPH radical of the obtained extracts varied significantly
(p < 0.001) from 17.75 ± 0.11 to 35.28 ± 1.19% according to the
extraction conditions used. Results showed that M. parviflora
leaf extract (1 mg GAE/mL) that was obtained at ethanol
(50%)/leaf powder ratio of 20:1 (v/w) or at a higher ratio using
an ethanol concentration of 60% exerted a high scavenging
activity against DPPH radicals that reached 76%. This finding
exceeded that obtained by Afolayan et al.[32] They found that
0.5 mg GAE/mL extract of M. parviflora. inhibited only 9.3% of
the DPPH radicals. Bouriche et al.[33] indicated that 89.03 lg/
mL of lyophilized M. parviflora leaf powder methanol extract
possessed the ability to scavenge 50% of the DPPH radicals.
The correlation between the total polyphenols content of
the extracts and their scavenging activity against DPPH radi-
cals is a significant (p < 0.001) negative correlation (r ¼
–0.457). This could be due to structure, concentration, and
the interaction between the antioxidants in the extract.[34]
To achieve regression models, linear, interactive (2 FI), quad-
ratic and cubic models were fitted to the experimental data and
the results are listed in Table 2. The data outlined in Table 2
reveal that, the most adequate model to represent experimental
data was quadratic model. As, the values of determination coef-
ficient (R2), adjusted determination coefficient (Adj R2) and pre-
dicated determination coefficient (Pred. R2) of quadratic model
were the highest and highly significant (p < 0.001) among other
models except cubic model, which was aliased or confounding.
Hence, the following second order polynomial equations with
interaction term were used to represent the relationship between
independent factors and responses in terms of coded factors.
 
YTPC mg GAE=100 g DW
¼ 1093:75 þ 32:50X1 þ 68:49X2  43:39X3  69:52X1 X2
þ 52:60X1 X3  53:00X2 X3  12:69X12  57:54X22  64:71X32
(4)
Yinhibition percentage of DPPH radical
¼ 21:29  0:58X1  3:91X2 þ 1:52X3  2:60X1 X2
 0:78X1 X3  0:17X2 X3 þ 2:54X12 þ 2:53X22  2:67X32
(5)
The ability of the obtained equations to describe the
response’s variability was determined using multiple regression
 PREPARATIVE BIOCHEMISTRY AND BIOTECHNOLOGY 5

Table 2. Adequacy of the model tested.

Source Std. Dev. R2 Adjusted R2 Predicted R2 PRESS Prob < F Remarks
Yield of total polyphenols (mg GAE/ 100 g dried leaf powder)
Linear 83.55 0.3582 0.3172 0.2277 3.948 Eþ005 0.0001
2FI 67.96 0.6025 0.5483 0.4585 2.768 Eþ005 <0.0001
Quadratic 49.00 0.8074 0.7652 0.6619 1.728 Eþ005 <0.0001 Suggested
Cubic 18.57 0.9744 0.9663 0.9498 25,654.86 <0.0001 Aliased
DPPH radical inhibition percentage of the extract
Linear 3.20 0.4718 0.4380 0.3482 593.90 <0.0001
2FI 2.99 0.5689 0.5101 0.3597 583.45 0.0288
Quadratic 1.91 0.8357 0.7996 0.7092 265.02 <0.0001 Suggested
Cubic 0.40 0.9933 0.9912 0.9873 11.59 <0.0001 Aliased
Quadratic models are underlined and the model is labeled as “Suggested” by the Design-Expert software. Suggestion is based on a subjective
scoring system that uses a combination of selected metrics to propose that quadratic model looks best compared with other models

Table 3. ANOVA analysis and statistical parameters of the model.

Yield of total polyphenols (mg GAE/ 100 g dried leaf powder) DPPH radical inhibition percentage of the extract
Source RC SS p Value RC SS p Value
Model 1093.75 4.13 Eþ05 < 0.0001 21.29 761.46 < 0.0001
X1 32.5 25,346.75 0.0023 –0.58 8.07 0.1448
X2 68.49 1.13 Eþ05 < 0.0001 –3.91 366.58 < 0.0001
X3 –43.39 45,175.07 < 0.0001 1.52 55.21 0.0004
X1 X2 –69.52 57,990.8 < 0.0001 –2.6 80.91 < 0.0001
X1 X3 52.6 33,206.38 0.0006 –0.78 7.23 0.1668
X2 X3 –53 33,708 0.0006 –0.17 0.35 0.759
X12 –12.69 2034.01 0.3628 2.54 81.18 < 0.0001
X22 –57.54 41,826.73 0.0002 2.53 80.59 < 0.0001
X32 –64.71 52,887.14 < 0.0001 –2.67 89.86 < 0.0001
Residual 98,447.47 149.74
Lack of fit 85,338.68 < 0.0001 143.67 < 0.0001
Pure error 13,108.79 6.07
Cor total 5.11 Eþ05 911.19
Std. dev. 49 1.91
Mean 1030.25 22.42
C.V.% 4.76 8.52
PRESS 1.73 Eþ05 265.02
R2 0.8074 0.8357
Adj R2 0.7652 0.7996
Pred R2 0.6619 0.7092
Adeq precision 13.059 19.359

analysis and analysis of variance (ANOVA) as outlined in
Table 3. The obtained models are highly significant, as its prob-
ability values are very low (p < 0.0001). Except regression coeffi-
cient of X12 for TPC and regression coefficients of X1 ; X1 X3 ;
and X2 X3 for DPPH radical inhibition percentage, all regression
coefficient are significant (p  0.0023). The values of R2 and
adjusted R2 are greater than 0.80 and 0.75, respectively, which
indicate the adequacy of the obtained models to predict the
experimental data.[27] The lack of fit (F-value) of 82.46 for TPC
and 299.92 for DPPH radical scavenging activity implies the
lack of fit is significant. There is only a 0.01% chance that a
“Lack of Fit F-value” this large could occur due to noise.
Adequate precision measures the signal to noise ratio. A ratio
larger than four is desirable. Similarly, adequate precision for
the responses (Table 3) are greater than 13, which also indicates
the adequacy of the signals.[35] As shown in Table 3, the CV of
the model is < 10, indicating that the model explains the
response adequately and indicates that the experimental values
are associated with a high degree of precision.[21]
Effect of process variables on the yield of polyphenols
and DPPH radical scavenging activity
Equations (4) and (5) were used to generate (3D) plots
(Figure 2) to illustrate the interactive effect of independent
variables (extraction time, solvent/leaf powder ratio (v/w)
and ethanol %) on the response variables. Generally, as
shown in Figure 2A–C, increasing extraction time and solv-
ent/leaf powder ratio (v/w) increased the yield of polyphe-
nols, while increasing the ethanol % above 50% decreased
the yield of polyphenols. The linear terms of extraction time
and solvent/leaf powder ratio (v/w) were significant
(p ¼ 0.0023 and p < 0.0001, respectively) and positive (Table
3), whereas its interactions and the quadratic term for solv-
ent/leaf powder ratio (v/w) only were significant
(p  0.0002) but negative. Hence, as appeared in Figure
2A–C, there is a curvilinear increase in the polyphenols yield
for all solvent/leaf powder ratios (v/w) used. On the other
hand, all terms of ethanol % were significant (p  0.0006)
and negative except the interaction term of ethanol % and
solvent/leaf powder ratio (v/w), which was positive. Thus,
there was a curvilinear decrease in the yield of polyphenols
for all ethanol concentrations used (Figure 2B,C).
Figure 2D–F illustrates the 3D surface plots of the DPPH
radical scavenging activity model. The interaction term of
extraction time, solvent/leaf powder ratio (v/w), and quad-
ratic term of extraction time were statistically significant
(p ˂ 0.0001) (Table 3), which result in a curvilinear change
in the inhibition percentage of DPPH radical for all extrac-
tion time investigated. Figure 2D shows that, the inhibition
6 E. A. ABD EL-SALAM AND N. F. S. MORSY

Figure 2. Response surface plots showing interaction effects of process variables (Extraction time (min), solvent/leaf powder ratio (mL/g) and ethanol %) on the
responses [TPC (mg GAE/100 g dried leaf powder) (A–C), inhibition percentage of DPPH radical (D-F)].

percentage of DPPH radical increased as extraction time
increased at low solvent/leaf powder ratio (v/w). However,
at high solvent/leaf powder ratio (v/w), the inhibition per-
centage of DPPH radical decreased as extraction time
increased. Except the interaction term of the solvent/leaf
powder ratio (v/w) and ethanol %, all terms of solvent/leaf
powder ratio (v/w) were statistically significant (p ˂ 0.0001).
The highest inhibition percentage of DPPH radical could be
observed at solvent/leaf powder ratio of 20:1 (v/w) (Figure 1D).
The linear and quadratic terms of ethanol % were significant
(p  0.0004), whereas its interactive terms were insignificant
(p  0.1668). The highest inhibition percentage of DPPH radical
could be obtained at an ethanol concentration of 50–55%
(Figure 1E,F). The optimum conditions to obtain Malva leaf
 PREPARATIVE BIOCHEMISTRY AND BIOTECHNOLOGY 7

Table 4. Identified phenolic and flavonoid compounds in the Malva leaf Conclusions
extract obtained at optimum conditions.
Compound lg/g leaf powder BBD was successfully used to optimize the extraction of pol-
Phenolic acids yphenols with high DPPH radical scavenging activity from
Gentisic acid 101.55 Malva parviflora L. leaves. Effect of independent variables
Rosmarinic acid 57.85
Vanillic acid 11.33 [ethanol concentration, and solvent/leaf powder ratio (v/w)]
Ferulic acid 3.99 on the responses was significant (p  0.0004). The absolute
Sinapic acid 86.44 errors between predicted and experimental values under
p-coumaric acid 371.86
Cinnamic acid 126.07 optimal conditions were low, which indicate the suitability
Flavonoids of the generated models to predict responses. The optimal
Apigenin-7-glucoside 346.28 extraction conditions were found to be extraction time
Catechin 35.78
Luteolin 271.66 15 min, solvent/leaf powder ratio 20:1 (v/w), and aqueous
Naringenin 557.33 ethanol concentration 53.40%. HPLC analysis indicated that
Apigenin 10.80 the extract obtained at optimal conditions was rich in narin-
Kaempferol 27.54
Chrysin 6.04 genin and p-coumaric acid. These findings indicate that M.
parviflora leaf extracts possess DPPH radical scavenging
activity and could be considered as a good source of natural
antioxidants.
extract with high DPPH radical scavenging activity were extrac-
tion time of 15 min, leaf powder/solvent ratio of 1: 20 (w/v),
and ethanol % of 53.40% with desirability of 0.742. DPPH rad- Disclosure statement
ical scavenging activity of the extract may be attributed to its
No potential conflict of interest was reported by the authors.
hydrogen donation potency. The antioxidant activity of each
phenolic compound depends on the number of hydroxyl
groups and their position in the structure. References
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