molecules
Article
Optimization of Extraction Process and Dynamic Changes in
Triterpenoids of Lactuca indica from Different Medicinal Parts
and Growth Periods
Junfeng Hao 1,2,3 , Qiang Si 1,2,3 , Zhijun Wang 1,2,3 , Yushan Jia 1,2,3 , Zhihui Fu 1,2,3 , Muqier Zhao 1,2,3 ,
Andreas Wilkes 4 and Gentu Ge 1,2,3, *
Citation: Hao, J.; Si, Q.; Wang, Z.; Jia,
Y.; Fu, Z.; Zhao, M.; Wilkes, A.; Ge, G.
Optimization of Extraction Process
and Dynamic Changes in
Triterpenoids of Lactuca indica from
Different Medicinal Parts and
Growth Periods. Molecules 2023, 28,
3345. https://doi.org/10.3390/
molecules28083345
Received: 27 February 2023
Revised: 28 March 2023
Accepted: 4 April 2023
Published: 10 April 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Molecules 2023, 28, 3345. https://doi.org/10.3390/molecules28083345
1 College of Grassland Resources and Environment, Inner Mongolia Agricultural University,
Hohhot 010019, China
2 Key Laboratory of Forage Cultivation and the Processing and High Efficient Utilization of the Ministry of
Agriculture, Inner Mongolia Agricultural University, Hohhot 010019, China
3 Key Laboratory of Grassland Resources of the Ministry of Education, Inner Mongolia Agricultural University,
Hohhot 010019, China
4 Values for Development Limited, Cambridge CB4 1RS, UK
* Correspondence: gegentu@163.com
Abstract: In this study, the triterpenoids in the leaves of Lactuca indica L.cv. Mengzao (LIM) were
extracted via microwave-assisted ethanol extraction, and the optimum extraction conditions for
triterpenoids were determined through single-factor experiments and the Box–Behnken method.
The effects of three factors (solid–liquid ratio, microwave power and extraction time) on the total
triterpenoids content (TTC) were evaluated. The TTC of different parts (roots, stems, leaves and
flowers) of LIM in different growth stages was studied, and the scavenging effects of the highest
TTC parts on DPPH, ABTS and hydroxyl free radicals were investigated. The results showed that
the optimum extraction conditions for microwave-assisted extraction of total triterpenoids from LIM
leaves were as follows: solid–liquid ratio of 1:20 g/mL; microwave power of 400 W; and extraction
time of 60 min. Under these conditions, the TTC was 29.17 mg/g. Compared with the fresh raw
materials, the TTC of the materials increased after freeze drying. The leaves of LIM had the highest
TTC, and the flowering stage was the best time. The triterpenoids from the leaves had a strong ability
to eliminate DPPH and ABTS free radicals, and the elimination effect of dried leaves was better than
that of fresh leaves, while the elimination effect of hydroxyl free radicals was not obvious. The tested
method was used to extract total triterpenoids from LIM using a simple process at low cost, which
provides a reference for developing intensive processing methods for L. indica.
Keywords: Lactuca indica L.cv. Mengzao; triterpenoids; microwave extraction; growth period; oxida-
tion resistance
1. Introduction
Lactuca indica L.cv. Mengzao (LIM) is an annual plant of the Compositae family. All
parts of Lactuca indica, including the roots, stems, leaves, flowers and seeds, have been used
as traditional Chinese medicines for thousands of years [1]. It has been widely used as
a kitchen herb for salads, sushi and soup, due to its delicate leaves and high nutritional
value [2]. It is fresh, juicy, slightly bitter, rich in flavonoids, lipids, terpenes, sterols and
other substances, and has biological effects such as heat clearing and detoxification, anti-
aging and anti-tumor effects, and anti-inflammatory, analgesic and antioxidant properties.
Among the plant contents, triterpenoids are compounds widely distributed in plants, and
have attracted increasing interest because of their antioxidant activity [3,4]. The main
triterpenoid compounds in L. indica are oleanolic-acid-type triterpenoids [5]. Triterpenoids
are compounds formed by the condensation of six isoprene monomers, which are impor-
tant natural chemical components. To date, most of the triterpenoids found have been
https://www.mdpi.com/journal/molecules
Molecules 2023, 28, 3345 2 of 15

tetracyclic triterpenoids and pentacyclic triterpenoids, which have many effects such as
tumor inhibition, liver protection and immunity enhancement [3,6].
Several studies have been conducted on extraction methods for total triterpenoids
in L. indica. Traditional extraction methods include reflux extraction [7] and Soxhlet ex-
traction [8], which are associated with long drying times, high energy consumption and
low efficiency. With the development of the concept of “green extraction” [9], various new
extraction technologies are increasingly being used to extract natural products from plants,
including microwave-assisted extraction [10], ultrasound-assisted extraction [11] and su-
percritical fluid extraction [12]. Among these extraction technologies, microwave extraction
is relatively popular and has some obvious advantages, e.g., low energy consumption,
short extraction time, higher extraction rate and lower cost [13]. To date, there have been
few reports on the extraction process and antioxidant activity of total triterpenoids from
L. indica, and also few reports on the distribution of total triterpenoids in different parts of
the plant and the best harvest time.
In this study, the raw material was LIM, a nationally approved variety independently
cultivated and bred by the team. On the basis of single-factor experiments, the effects
of the solid–liquid ratio, microwave power and extraction time on total triterpenoids in
LIM leaves were investigated to determine the best extraction conditions. On this basis,
the distribution of and dynamic changes in the TTC in various parts (roots, stems, leaves
and flowers) of LIM in different growth periods were investigated, the optimal utilization
part and harvest time were determined, and its abilities to eliminate DPPH free radicals,
hydroxyl free radicals and ABTS free radicals were investigated. It is expected that this
research will provide a theoretical basis and reference for further development of diversified
and intensive processing of L. indica.

2. Results and Discussion

2.1. Optimization of TTC Conditions
2.1.1. Influence of Solid–Liquid Ratio on TTC
The solid–liquid ratio is a key factor for an efficient recovery of biomolecules from dif-
ferent materials, since the extraction of these compounds is influenced by the mass transfer
resistance associated with the solid matrix structure. Moreover, in the face of a subsequent
industrial scale-up, a compromise must be reached between a sufficient solid–liquid ratio
to achieve a suitable extraction yield that is low enough to reduce the energy costs [14].
In this study, as the solid–liquid ratio increased from 1:5 to 1:20 g/mL, the TTC increased
sharply (Figure 1a). When the solid–liquid ratio increases, the cavitation effect generated
by microwaves will be stronger, and the mass transfer process will be faster, resulting
in a higher extraction efficiency [15]. However, when the solid–liquid ratio increased to
1:20 g/mL, the effective components were basically dissolved by a certain proportion of the
solvent, so the extraction rate stopped increasing. Furthermore, if the amount of ethanol is
too high, the dissolution of other impurities will also increase correspondingly, resulting in
a decrease in the extraction volume of triterpenoids [16]. Therefore, from the perspective of
the extraction volume, solvent consumption and production cost, a solid–liquid ratio of
1:15~1:25 g/mL was selected for further optimization.

2.1.2. Influence of Microwave Power on TTC

Microwave-assisted extraction is one of the most commonly used extraction methods.
Microwave heating can improve the diffusion ability of target components, thereby short-
ening extraction time [17]. This showed that the microwave power has a certain impact on
the TTC (Figure 1b). The TTC reached a maximum at 400 W, and then showed a downward
trend. This result is consistent with previous reports also showing that the TTC increased
with increasing microwave power within a certain microwave power range [18,19]. When
the microwave power is too high, the solvent volatilizes. Therefore, a microwave power of
300~500 W was selected for further optimization.
Molecules 2023, 28, x FOR PEER REVIEW 3 of 16
Molecules 2023, 28, 3345 3 of 15

(a) (b)
35 35

Extraction yield /(mg/g)

Extraction yield /(mg/g)

28 28

21 21

14 14

7 7

0 0
1:5 1:10 1:15 1:20 1:25 200 300 400 500 600
Solid–liquid ratio /(g/mL) Microwave power /W

(c) 35
Extraction yield /(mg/g)

28

21

14

0
15 30 45 60 75
Extraction time /min

Figure
Figure1.1.The
Theeffects
effectsofofthe
thesolid–liquid
solid–liquidratio
ratio(a),
(a),microwave
microwavepower
power(b)
(b)and
andextraction
extractiontime
time(c)(c)on
on
TTC extraction from LIM leaves.
TTC extraction from LIM leaves.

2.1.2.
2.1.3.Influence of Microwave
The Influence Power
of Extraction on TTC
Time on TTC
Microwave-assisted
The choice of extraction extraction
time was is one of the
another most commonly
important used extraction
step to guarantee meth-
the distribution
ods. Microwave
equilibrium heating can improve
of triterpenoids betweenthe thediffusion
sample and ability of targetsolvent
extraction components,
[20]. The thereby
results
shortening
are shownextraction
in Figure time [17].
1c. As theThis showed
extraction thatincreased
time the microwave
from 15power
min tohas
60amin,
certain
the im-
TTC
pact on the significantly.
increased TTC (Figure 1b). AfterThe60 TTC
min, reached
the TTCabeganmaximum at 400With
to decline. W, and then showed
a longer a
extraction
duration, the
downward trend.TTCThis
increased first
result is and thenwith
consistent decreased.
previous The reasonalso
reports may be that that
showing at thethe
be-
ginning
TTC of extraction,
increased the extraction
with increasing microwavevolumepowerincreased
within due to themicrowave
a certain different concentrations
power range
of rawWhen
[18,19]. materials the and solvents.
microwave Afterisa too
power certain
high,duration, the volatilizes.
the solvent mass concentration
Therefore, ofaactive
mi-
substances
crowave power inside and outside
of 300~500 W was theselected
raw material reached
for further a relative balance, and the triter-
optimization.
penoids in the raw material were no longer easily leached, so the extraction volume tended
to stabilize.
2.1.3. In addition,
The Influence the extraction
of Extraction of triterpenoids
Time on TTC will be affected by the dissolution of
other
The alcohol-soluble substances
choice of extraction time wasduring the extraction
another importantperiod
step to[21], resulting
guarantee theindistribu-
leaching
tion equilibrium of triterpenoids between the sample and extraction solvent [20]. min
of impurities, thus reducing the TTC. After comprehensive consideration, 45~75 was
The re-
selected for further optimization.
sults are shown in Figure 1c. As the extraction time increased from 15 min to 60 min, the
TTC increased significantly. After 60 min, the TTC began to decline. With a longer extrac-
2.2. Optimization of Microwave-Assisted Parameters for Triterpenoids by BBD
tion duration, the TTC increased first and then decreased. The reason may be that at the
2.2.1. Model Fitting Analysis
beginning of extraction, the extraction volume increased due to the different concentra-
tions ofAfter
rawthese pre-experiments,
materials and solvents. three
After main variable
a certain levelsthe
duration, were
mass determined for of
concentration the
solid–liquid ratio (1:15~1:25 g/mL), microwave power (300~500 W) and extraction
active substances inside and outside the raw material reached a relative balance, and the time
(45~75 min).in
triterpenoids The
theexperimental
raw materialdesign and
were no corresponding
longer response
easily leached, data
so the for the TTC
extraction from
volume
LIM are presented in Table 1. Regression analysis showed that the extraction of the TTC
tended to stabilize. In addition, the extraction of triterpenoids will be affected by the dis-
was predicted by the second-order equation (Equation (1))
solution of other alcohol-soluble substances during the extraction period [21], resulting in
leaching of impurities, thus reducing the TTC. After comprehensive consideration, 45~75
Y = 32.94 + 0.28A
min+ was
0.60B − 0.39C
selected for+ further + 1.24AC − 0.40BC − 2.05A2 − 2.72B2 − 2.05C2
0.77AB optimization. (1)
Molecules 2023, 28, 3345 4 of 15

where Y represents the response of the TTC, A represents the solid–liquid ratio, B represents
the microwave power and C represents the extraction time.

Table 1. Box–Behnken program and experimental results.

Solid–Liquid Ratio Microwave Power Extraction Time Response of TTC

Test
/(g/mL) /(W) /(min) /(mg/g)
1 1:15 300 60 27.66
2 1:25 300 60 26.99
3 1:15 500 60 27.81
4 1:25 500 60 30.20
5 1:15 400 45 30.64
6 1:25 400 45 28.43
7 1:15 400 75 26.76
8 1:25 400 75 29.52
9 1:20 300 45 27.58
10 1:20 500 45 28.90
11 1:20 300 75 28.03
12 1:20 500 75 28.16
13 1:20 400 60 32.67
14 1:20 400 60 32.55
15 1:20 400 60 33.10
16 1:20 400 60 32.94
17 1:20 400 60 33.44

Table 2 shows the results of calculating the regression coefficients for the intercept,
and linear, quadratic and interactive terms of the model. Significance testing (p < 0.001)
of the regression equations showed that the quadratic multiple regression model was
extremely significant. The independent variables (A, B, C) and the three quadratic terms
(A2 , B2 , C2 ) of the equation had a significant impact on the TTC (p < 0.05). There was also
a significant interaction between the solid–liquid ratio and microwave power (AB), and
between the solid–liquid ratio and extraction time (AC) (p < 0.05). For each item in the
model, a larger F value and a smaller p value indicate a more significant impact on the
corresponding response variables [22]. From the results of the variance analysis in Table 2,
the decision coefficient (R2 ) of the model was 0.98, and the adjusted decision coefficient
(Adj-R2 ) was 0.96, indicating that the prediction results had a strong correlation with the
actual results [23]. The model p value was less than 0.05, indicating that the experimental
model was significant. The mismatch was not significant (p = 0.1129 > 0.05), indicating
that the model fit the actual data well. The F values showed that the order of influence of
various factors on the TTC was as follows: microwave power (B) > extraction time (C) >
solid–liquid ratio (A).

2.2.2. Model Applicability Diagnosis

The applicability of the linear regression simulation of the total triterpenoids extracted
from the leaves of LIM was diagnosed using the three diagnostic charts for the residual
normal probability distribution, the studentized residuals and the predicted and measured
values. The results are shown in Figure 2. It can be seen from the residual normal probability
distribution diagram in Figure 2a that the distribution of most data points was concentrated
together, and was close to a straight line, indicating that the model was stable and consistent
with a normal distribution. According to the studentized residual diagram in Figure 2b, all
data points were distributed within the range of ±3, indicating that the model was in good
agreement with the total triterpenoids extracted from LIM. It can be seen from Figure 2c
that the data points of the predicted values and the actual values were close to a straight
line, and the model fit well across the whole regression area. The model can effectively
predict the total triterpenoid extraction amount in the leaves of LIM [10].
Molecules 2023, 28, 3345 5 of 15

Table 2. ANOVA for the response surface model.

Source Sum of Squares Df Mean Squares F Value p Value

Model 87.9 9 9.77 35.78 <0.0001
Solid–liquid ratio 0.64 1 0.64 2.34 0.0317
Microwave power 2.89 1 2.89 10.59 <0.0001
Extraction time 1.19 1 1.19 4.35 0.0017
AB 2.35 1 2.35 8.6 0.0219
AC 6.18 1 6.18 22.65 0.0021
BC 0.36 1 0.36 1.32 0.2884
A2 17.76 1 17.76 65.06 <0.0001
B2 31.23 1 31.23 114.41 <0.0001
C2 17.67 1 17.67 64.73 <0.0001
Residual 1.91 7 0.27
Lack of fit 1.42 3 0.47 3.85 0.1129
Pure error 0.49 4 0.12
Molecules 2023, 28, x FOR PEER REVIEW Cor total 89.81 16 6 of 16
R2 87.9 9 9.77 35.78 <0.0001
Adj-R2 0.64 1 0.64 2.34 0.0317

Figure 2. Diagnostic plots for model adequacy for extraction of TTC. (a) Residual normal probability
Figure Diagnostic residuals;
plot; (b)2.studentized plots for model adequacy
(c) predicted and for extraction
actual of TTC.
values. The data(a) Residual
points normal probability
with different colors
plot;
in the(b) studentized
figure representresiduals; (c) predicted
the test groups and actual
with different TTCs.values. The data points with different colors
in the figure represent the test groups with different TTCs.
2.2.3. Response Surface Optimization Analysis of Triterpenoid Extraction Conditions
The influence of variables and their interactions on the TTC is illustrated through an
inspection of the three-dimensional (3D) response surfaces and two-dimensional (2D)
contour maps (Figure 3a–f). The interaction between variables can be interpreted from the
shape of the contour map. An elliptic contour map shows that the interaction between the
Molecules 2023, 28, 3345 6 of 15

2.2.3. Response Surface Optimization Analysis of Triterpenoid Extraction Conditions

The influence of variables and their interactions on the TTC is illustrated through
an inspection of the three-dimensional (3D) response surfaces and two-dimensional (2D)
contour maps (Figure 3a–f). The interaction between variables can be interpreted from the
shape of the contour map. An elliptic contour map shows that the interaction between
the corresponding variables was significant, while a circular contour map shows that the
interaction between the corresponding variables was not significant [24]. The maximum
predicted response was located at the peak of the three-dimensional response surface,
and the corresponding point was defined by the minimum ellipse in the two-dimensional
contour map. The steepness of the surface can reflect the impact of the investigated factors
on the response value. The greater the steepness, the greater the impact on the response
value [25]. In this study, the opening of the 3D response surface was downward. With an
increase in two factor values (AB, AC, BC), the response value of the TTC will also increase.
After the response value increased to the extreme value, it gradually decreased with the
increase in two factor values.
Figure 3a,b show the comprehensive effects of the solid–liquid ratio and microwave
power on the TTC. It is clearly shown that when the solid–liquid ratio was increased to
1:20 g/mL and the microwave power increased to 400 W, the TTC increased and reached
its peak. As the two factors further increased, the TTC gradually decreased. The results
showed that only a proper solid–liquid ratio and a certain extraction power could signifi-
cantly improve the TTC, and too low or too high values of these factors would lead to a
decrease in extraction yield. In addition, the contour map was elliptical, indicating that the
interaction between the ultrasonic power and extraction time was significant [26].
Figure 3c,d show the comprehensive effect of the solid–liquid ratio and extraction
time on the TTC. When the microwave power remained unchanged, the TTC increased
with increasing solid–liquid ratio (1:15~1:20 g/mL) and extraction time (45~60 min), and
reached a peak in the middle stage. As the solid–liquid ratio and extraction time increased,
the TTC began to decrease, which may be due to the dissolution of other alcohol-soluble
substances after a long extraction period in the presence of more solvents. In addition,
the elliptical shape of the contour map indicated a significant interaction between these
two factors.
Figure 3e,f show the combined effect of the microwave power and extraction time on
the TTC. Increasing the microwave power to 400 W led to an increase in the TTC, but a
further increase in the microwave power led to a slow decrease in the TTC. For the effect
of the extraction time on the TTC, the TTC reached its maximum after about 60 min and
then decreased slightly, which may be due to the decomposition of triterpenoids with the
increased extraction time. The circular contour map shows that the interaction between
these two parameters was not significant.
The results of the optimization using Design-Expert 8.0.6 analysis software showed
that the best extraction conditions for the TTC were a solid–liquid ratio of 1:20.33 g/mL,
microwave power of 412.44 W and extraction time of 58.75 min. Under this condition,
the predicted value of the TTC in LIM leaves was 29.05 mg/g. In order to ensure that the
predicted value did not deviate from the actual results, the best conditions derived were
verified through experiments. Based on the operability of the test, the best parameters
for prediction were fine-tuned, i.e., solid–liquid ratio of 1:20 g/mL, microwave power
of 400 W and extraction time of 60 min. Three parallel validation experiments were con-
ducted under this condition, and the average extraction yield of total triterpenoids was
29.17 ± 0.15 mg/g, which verified the accuracy of the RSM model. The reliability of the
optimized process parameters was fully explained.
Molecules2023,
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Figure 3. Three-dimensional response surface plots showing the effects of different parameters on
Figure 3. Three-dimensional response surface plots showing the effects of different parameters on
TTC. Solid–liquid ratio and ultrasonic power (a); solid–liquid ratio and ethanol concentration (b);
TTC. Solid–liquid ratio and ultrasonic power (a); solid–liquid ratio and ethanol concentration (b);
solid–liquid ratio and extraction time (c); ultrasonic power and ethanol concentration (d); ultrasonic
solid–liquid ratio and extraction
power and extraction time (c);concentration
time (e); ethanol ultrasonic power and ethanol
and extraction concentration
time (f). (d); ultrasonic
power and extraction time (e); ethanol concentration and extraction time (f).
Figure 3a,b show the comprehensive effects of the solid–liquid ratio and microwave
power on the TTC. It is clearly shown that when the solid–liquid ratio was increased to
1:20 g/mL and the microwave power increased to 400 W, the TTC increased and reached
Molecules 2023, 28, 3345 8 of 15

2.3. Dynamic Changes in TTC in Different Parts of LIM in Different Growth Stages
The TTC extraction test was applied to fresh and dried samples of different parts of
LIM harvested in different growth stages using the optimized extraction process. The TTC
of the roots, stems, leaves, flowers and the whole plant of LIM harvested in different growth
stages was measured three times in parallel, as shown in Figure 4. From the perspective of
sample treatments, compared with the fresh raw materials, the TTC of the whole plant and
of each plant part increased after freeze-drying. Among the different parts, the increase
in the TTC in the leaves and flowers was the most obvious. Due to the high moisture
content of the fresh samples and low TTC, the TTC of each part showed an increasing trend
with the loss of moisture during the drying process [27]. In addition, triterpenoids are
secondary metabolites of plants, which are usually the products of environmental stresses
such as water and temperature, and the drying process is a drought stress process for
freshly harvested plants, which may induce the formation of triterpenoids and an increase
in the relevant active components [28]. In this study, the TTC of each part of LIM increased
significantly after drying, which may be due to the formation or increased content of
triterpenoids induced by drought stress.
The content of active substances in L. indica varies during the plant growth process.
Dong [29] pointed out that the TTC in L. indica showed a downward trend before flowering,
and then increased sharply to reach a maximum value. The research results of Wang [30]
showed that the seedling stage was the harvesting stage with the highest TTC in L. indica.
In this experiment, the total triterpenoids were extracted from fresh samples of LIM in
different periods. The results showed that the TTC in the initial flowering stage was the
highest, after which a downward trend was observed. The freeze-dried plants also returned
similar results, indicating that the initial flowering period is the most suitable period for
the extraction of total triterpenoids in LIM. In addition, the TTC of different parts of LIM
showed dynamic changes during the growth process.
Figure 4e,f show that the leaves had the highest TTC in all the fresh and dried samples.
Figure 4e shows that the extraction yield of fresh leaf samples in the middle flowering
stage was the highest, reaching as high as 19.19 mg/g. The yield of dried leaf samples
extracted in the middle flowering stage was the highest, reaching 33.96 mg/g. Among
the fresh samples, the extraction yield of the roots in the seeding stage was the lowest, at
1.82 mg/g. Among the dried samples, the extraction yield of the samples extracted from
the whole plants in the filling stage was the lowest, at 5.39 mg/g. As the main organ for
transporting nutrients, the stems provide a pathway for the synthesis and transportation of
total triterpenoids to the roots, leaves and flowers [4]. Leaves, as the main organ for the
accumulation of effective components, accumulate a relatively high TTC. Therefore, leaves
should be considered the main plant part targeted for the extraction of triterpenoids in LIM.
In order to study the antioxidant activity of the total triterpenoids of LIM, fresh and dried
leaf samples in the middle flowering stage were selected to test their antioxidant activity.

2.4. Antioxidant Activity of Total Triterpenoids in LIM

DPPH is a purple stable free radical. It can absorb light at 517 nm. After receiving
the hydrogen atom of an antioxidant, it changes from purple to yellow. After adding
a certain antioxidant, some free radicals are removed, and the absorption intensity at
this wavelength is weakened, so it is often used to evaluate the antioxidant properties
of substances [31]. In this study, taking L-ascorbic acid as the positive control, the total
triterpenoids in the leaves of LIM showed a strong scavenging effect on DPPH, and the
scavenging ability was positively correlated with the concentration of total triterpenoids
(Figure 5a). In the concentration range of 0.1~0.6 mg/mL, the scavenging ability of DPPH
increased significantly with increasing total triterpenoid concentration in the leaves of LIM.
The scavenging effect of fresh and dried leaves of LIM on DPPH was lower than that of
L-ascorbic acid, and their IC50 values were 0.21 mg/mL and 0.28 mg/mL, respectively,
indicating that drying fresh leaves of LIM can enhance the scavenging effect on DPPH free
radicals.
Molecules 2023, 28, x FOR PEER REVIEW 9 of 16
Molecules 2023, 28, 3345 9 of 15

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r we Fil ed ge we we
r we Fil
Ve flo flo flo Se Ve flo flo flo
itial d le eak itial d le eak
In id P In id P
M M

Figure 4. Analysis
Figure of TTC
4. Analysis fromfrom
of TTC freshfresh
Lactuca indica indica
Lactuca samples (a)—roots;
samples (c)—stems;
(a)—roots; (e)—leaves;
(c)—stems; (g)—
(e)—leaves;
flowers; (i)—whole plants) and dried Lactuca indica samples (b)—roots; (d)—stems; (f)—leaves;
(g)—flowers; (i)—whole plants) and dried Lactuca indica samples (b)—roots; (d)—stems; (f)—leaves;
(h)—flowers; (j)—whole plants).
(h)—flowers; (j)—whole plants).
Molecules 2023, 28, 3345 10 of 15

Figure 5b shows that L-ascorbic acid, dried leaves and fresh leaves of LIM with
different mass concentrations had a certain elimination effect on ABTS+ free radicals. In
the concentration range of 0.1~0.6 mg/mL, the scavenging effect of dried leaves and fresh
leaves on ABTS free radicals increased with increasing TTC in the extracts. The scavenging
effect of 0.6 mg/mL was the strongest in the dried and fresh leaves, with scavenging rates
of 72.99% and 55.51%, respectively, which were weaker than those of L-ascorbic acid, and
their IC50 values were 0.18 mg/mL and 0.32 mg/mL, respectively. These results showed
that drying fresh leaves of LIM enhanced the TTC scavenging effect on ABTS+ free radicals.
A reaction system model was established according to the Fenton reaction method,
and the mixture of H2 O2 and Fe2+ was used to generate hydroxyl. Then, salicylic acid was
added to produce colored products. The product has strong absorption at the 510 mm
wavelength. If an antioxidant is added to the reaction system, it will compete with salicylic
acid for hydroxyl and reduce the amount of colored products [32]. In this study, a series
of extracts of dried and fresh leaves of LIM with different concentrations were added to
the reaction system. By measuring the absorbance of each concentration at the 510 nm
wavelength, the scavenging effects of the tested substances on hydroxyl radicals were
detected. Figure 5c shows that the total triterpenoids in the fresh and dried leaves increased
the scavenging effect on hydroxyl radicals as the concentration of the extract increased. The
scavenging effect of both fresh and dried leaves on hydroxyl radicals was weaker than that
of L-ascorbic acid, and their IC50 values were 0.36 mg/mL and 0.74 mg/mL, respectively.
Molecules 2023, 28, x FOR PEER REVIEW 11 of 16
This showed that drying fresh leaves of LIM enhanced the scavenging effect of the leaves
on ABTS free radicals.

Figure
Figure5.5.Antioxidant
Antioxidant activities
activities (a)—DPPH radical elimination
(a)—DPPH radical eliminationability;
ability;(b)—ABTS
(b)—ABTSradical
radical elimina-
elimination
tion
ability; (c)—hydroxyl radical elimination ability) of total triterpenoids in fresh and driedleaves
ability; (c)—hydroxyl radical elimination ability) of total triterpenoids in fresh and dried leaves
of LIM.
of LIM.

Figure 5band
3. Materials shows that L-ascorbic acid, dried leaves and fresh leaves of LIM with dif-
Methods
ferent massMaterials
3.1. Plant concentrations had a certain elimination effect on ABTS+ free radicals. In the
concentration rangewere
LIM samples of 0.1~0.6 mg/mL,
collected the planting
from the scavenging
baseeffect of dried
of Inner leavesAgricultural
Mongolia and fresh
leaves on ABTS free radicals increased with increasing TTC in the extracts. The scavenging
University from June to September 2019. The collected samples were divided into different
effect of 0.6 mg/mL was the strongest in the dried and fresh leaves, with scavenging rates
of 72.99% and 55.51%, respectively, which were weaker than those of L-ascorbic acid, and
their IC50 values were 0.18 mg/mL and 0.32 mg/mL, respectively. These results showed
that drying fresh leaves of LIM enhanced the TTC scavenging effect on ABTS+ free radi-
cals.
Molecules 2023, 28, 3345 11 of 15

parts, i.e., roots, leaves, stems and flowers. All samples were prepared in two ways, fresh
and dried. The fresh samples were processed for 2 min by crushing the sample with an
RCD-1A homogenizer (Jiangsu Dongpeng Instrument Manufacturing Co., Ltd., Changzhou,
China), while the dried samples were vacuum freeze-dried for 48 h using a Christ ALPHA
1-4LDplus lyophilizer (Marin Christ, Osterode, Germany) and then crushed into a uniform
powder (40 mesh) for analysis.

3.2. Chemicals and Reagents

Oleanolic acid, L-ascorbic acid, 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) and
2,20 -diazo-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) were all purchased from
Shanghai Yuanye Technology Co. Ltd. (Shanghai, China). Hydrogen peroxide, ferrous
sulfate, salicylic acid and ethanol were all purchased from Sinopharm Chemical Reagent
Co. Ltd. (Shanghai, China). All chemicals and reagents were of analytical grade.

3.3. Microwave-Assisted Extraction of Total Triterpenoids

Total triterpenoid extraction was performed using an XH-100B series microwave
extraction instrument (Beijing Xianghu Science and Technology Development Co. Ltd.,
Beijing, China). Freeze-dried samples (crushed through a 40-mesh sieve) and fresh sample
fragments (cut to a particle size of about 1 mm) of LIM leaves were added into a conical
flask with a stopper, and 70% ethanol solution [30] was added to achieve the target ratio of
material to liquid. The sample solution was extracted under different microwave powers
and treatment times before centrifuging (15,000 rpm, 15 min) and extracting the supernatant.
The obtained solution was diluted to 50 mL with 50% ethanol solution by volume fraction,
and then shaken to obtain the triterpenoid extracts from the leaves of LIM. Samples were
stored at 4 ◦ C. Each experimental treatment was repeated three times.

3.4. Determination of TTC

An amount of 25 mg of oleanolic acid was weighed and fully dried at 105 ◦ C. The
solution was dissolved and diluted with methanol into a 25 mL volumetric flask, shaken to
make it completely dissolve and prepared as a reference solution with a mass concentration
of 1.0 mg/mL. Amounts of 0, 0.2, 0.4, 0.6, 0.8 and 1 mL of the oleanolic acid reference
solution with a mass concentration of 1.0 mg/mL were placed into different test tubes.
After completely volatilizing the solvent at room temperature, 1.6 mL of perchloric acid
solution and 0.4 mL of 5% vanillin–acetic acid solution were added to each test tube,
heated in a 70 ◦ C water bath for 15 min and cooled naturally, and then 5 mL of glacial
acetic acid was added and shaken well. The absorbance of the reference solution of
different concentrations at the wavelength of 548 nm was determined with a UV-1900i
series ultraviolet–visible spectrophotometer (Shimadzu Co. Ltd., Kyoto, Japan), and a blank
test was performed. With different concentrations of oleanolic acid as the abscissa and
absorbance A as the ordinate, we drew the standard curve. The standard curve equation
was y = 0.0862x + 0.0012, R2 = 0.9999.
An amount of 1 mL of total triterpene extracts of LIM was taken, and the concentration
of total triterpenes in the LIM sample solution was measured according to the method of
the standard curve equation.

3.5. Single-Factor Experiment

The effects of the solid–liquid ratio, microwave power and extraction time on the TTC
from the leaves of LIM were studied by changing the level of one factor and keeping the
other two factors constant. The detailed conditions for each extraction were as follows:
(1) when the liquid–solid ratios were 5, 10, 15, 20 and 25 g/mL, the samples were extracted
with an ethanol concentration of 70% at 300 W for 30 min; (2) when the microwave power
settings were 200, 300, 400, 500 and 600 W, the samples were extracted with a liquid–solid
ratio of 10 g/mL and an ethanol concentration of 70% for 30 min; (3) when the extraction
Molecules 2023, 28, 3345 12 of 15

times were 15, 30, 45, 60 and 75 min, the samples were extracted with a solid–liquid ratio of
10 g/mL and an ethanol concentration of 70% at 300 W.

3.6. Optimization Experimental Design

Based on the preliminary results of the single-factor test, the proper range for each
factor was determined, and then response surface methodology (RSM) was conducted
using BBD in Design Expert 8.0.6 software (StatEase® , Minneapolis, MN, USA). As shown
in Table 3, the three factors chosen for this study were designed as A (solid–liquid ratio),
B (microwave power) and C (extraction time) and were prescribed three levels, coded as
−1, 0 and 1. The TTC was the response value. The experimental design included 19 trials,
including 5 replicates of the center point.

Table 3. Box–Behnken design factors and levels.

Level
Factor
−1 0 1
A: Solid–liquid ratio/(g/mL) 15:1 20:1 25:1
B: Microwave power/W 300 400 500
C: Extraction time/min 45 60 75

3.7. Determination of Antioxidant Activity of MAE Extracts

3.7.1. DPPH Radical Scavenging Assay
The DPPH radical scavenging assay of the sample extracts was performed as described
in a previous study with slight modifications [33]. An amount of 2 mL of sample extraction
solution with different mass concentrations (0.1~0.6 mg/mL) was mixed with 2 mL of
0.1 mg/mL freshly prepared DPPH solution (in methanol). After incubation in the dark for
30 min at room temperature, the absorbance (Ai ) at 517 nm was read against a blank using
a UV-1900i series ultraviolet–visible spectrophotometer. The DPPH scavenging activity
was calculated using the formula

Elimination rate(%) = [1 − (Ai − Aj )/AO ] × 100% (2)

where Ao is the absorbance of the control group (70% ethanol solution instead of sample
solution), and Aj is the absorbance of the negative control (methanol instead of DPPH
solution). L-ascorbic acid (0.1~0.6 mg/mL) was used as the reference. The semi-inhibitory
concentration (IC50 ) value, the sample concentration required to quench 50% of DPPH
radicals, was obtained by fitting the polynomial using Statistical Product and Service
Solutions 14.0 (SPSS Inc., Chicago, IL, USA). The lower the IC50 value, the stronger the free
radical scavenging ability of the antioxidant.

3.7.2. ABTS Radical Scavenging Assay

The ABTS radical scavenging assay applied to the sample extracts was performed
as described in a previous study with slight modifications [34]. With L-ascorbic acid as
the positive control, the absorbance of the sample extracts with different concentrations
(0.1~0.6 mg/mL) was determined at 734 nm using an ultraviolet spectrophotometer. The
ABTS radical scavenging activity was calculated according to Equation (2), where Ao repre-
sents the absorbance of the control group (70% ethanol solution instead of sample solution),
Ai represents the absorbance of the sample solution and Aj represents the absorbance of
the sample only (methanol instead of ABTS solution).

3.7.3. Hydroxyl Radical Scavenging Activity

The hydroxyl radical scavenging assay for the sample extracts was performed as
described in a previous study with slight modifications [35]. An amount of 2 mL of
6 mmol/L FeSO4 solution was mixed with 2 mL of extraction solution with different mass
Molecules 2023, 28, 3345 13 of 15

concentrations (0.1~0.6 mg/mL), and 2 mL of 6 mmol/L H2 O2 solution was added to

the mixed solution and left to stand for 10 min. Then, 2 mL of 6 mmol/L salicylic acid
solution was added and left to stand for 30 min. The hydroxyl radical scavenging rate was
computed according to Equation (2), where Ai , Aj and Ao correspond to the absorbances of
the sample solution reaction, reagent blank (without DPPH solution) and control (without
the sample), respectively, all of which were measured at 510 nm.

3.8. Statistical Analysis

Data are expressed as the mean ± standard deviation of triplicate measurements.
Analysis of variance was performed using SPSS 24.0 (SPSS Inc., Chicago, IL, USA). Statistical
analysis of the results was carried out using analysis of variance (ANOVA) with Duncan’s
test, and the statistical significance of differences between the means of each factor was
determined at p < 0.05. Charts were drawn with OriginPro 2021 (Origin Lab® , Northampton,
MA, USA).

4. Conclusions
Our method provides information on the complete extraction and antioxidant deter-
mination of triterpenoids in LIM. In this study, three-variable, three-stage experiments
conducted using BBD and RSM were applied to investigate the microwave-assisted ethanol
aqueous solution extraction method to determine the highest extraction rate of triterpenoids
in LIM leaves. Furthermore, the differences in total triterpenoids in different parts of LIM
harvested in different growth periods were analyzed. These data not only show that there
were differences in the active components of the aboveground and underground parts of
LIM, but also provide an effective reference for the determination of the best harvest time
of different plant parts. The leaves are the main part to harvest, and the flowering stage is
the best harvest time. Our study also shows that the total triterpenoids in the leaves have a
high antioxidant potential in vitro, with potential applications in food, feed and nutrients.
In the future, in vitro digestibility, bioavailability, toxicology and animal research is needed
to develop leaves into commercial ingredients.

Author Contributions: Conceptualization, J.H., Y.J. and G.G.; methodology, J.H. and Z.W.; experimen-
tation, J.H.; analysis, M.Z., Q.S. and Z.F.; writing—original draft preparation, J.H.; writing—review
and editing, A.W. and G.G.; funding acquisition, G.G. All authors have read and agreed to the
published version of the manuscript.
Funding: This work was supported by the Inner Mongolia Autonomous Region Natural Science
Fundation (No. 2021MS03095).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Sun, X.Y.; Sun, F.Y. Shennong Bencao Jing; The Commercial Press: Beijing, China, 1995.
2. Oliya, B.K.; Kim, M.Y.; Lee, S.-H. Development of genic-SSR markers and genetic diversity of Indian lettuce (Lactuca indica L.) in
South Korea. Genes Genom. 2018, 40, 615–623. [CrossRef] [PubMed]
3. Li, K.W.; Liang, Y.Y.; Xie, S.M.; Niu, F.J.; Guo, L.Y. Ixeris sonchifolia: A review of its traditional uses, chemical constituents,
pharmacology and modern applications. Biomed. Pharmacother. 2020, 125, 109869. [CrossRef] [PubMed]
4. Hao, J.; Li, Y.; Jia, Y.; Wang, Z.; Rong, R.; Bao, J.; Zhao, M.; Fu, Z.; Ge, G. Comparative Analysis of Major Flavonoids among Parts
of Lactuca indica during Different Growth Periods. Molecules 2021, 26, 7445. [CrossRef]
5. Liu, S.M.; Xie, W.D.; Meng, F.J. Research progress on chemical constituents and pharmacological activities of Ixeris. Lishizhen Med.
Mater. Med. 2010, 21, 975–977.
6. El-Mekkawy, S.; Meselhy, M.R.; Nakamura, N.; Tezuka, Y.; Otake, T. Anti-HIV-1 and anti-HIV-1-protease substances from
Ganoderma lucidum. Phytochemistry 1998, 49, 1651. [CrossRef] [PubMed]
Molecules 2023, 28, 3345 14 of 15

7. Wang, J.; Zhao, L.X.; Lei, J.D.; He, J.; Guo, L.Q. Study on optimization of extraction technology of ursolic acid from Eriobotrya
japonica leaves by response surface-method and its purification. Chin. J. Trop. Crops 2015, 36, 1518–1524.
8. Guo, J.; Wang, L.L.; Wu, X.S.; Liu, T.G.; Wu, J.S. Ultrasonic extraction of ursolic acid from xishan Jujube (zizyphus jujuba Mill).
Chin. Agric. Sci. Bull. 2016, 32, 175–181.
9. Chemat, F.; Vian, M.A.; Cravotto, G. Green extraction of natural products: Concept and principles. Int. J. Mol. Sci. 2012, 13,
8615–8627. [CrossRef]
10. Fu, Y.Y. Microwave and enzyme co-assisted aqueous two-phase extraction of polyphenol and lutein from marigold (Tagetes erecta
L.) flower. Ind. Crops Prod. 2018, 123, 296–302. [CrossRef]
11. Chen, X.Q.; Liu, L.L.; Sun, T.T.; Zhang, Y. Optimization of extraction of total phenolics and total flavonoids from Phellodendron
amurense fruit by response surface method and their antioxidant activities. Fine Chem. 2020, 37, 300–308.
12. Klejdus, B.; Lojková, L.; Lap?Ík, O.; Koblovská, R.; Moravcová, J.; Kubáň, V. Supercritical fluid extraction of isoflavones from
biological samples with ultra-fast high-performance liquid chromatography/mass spectrometry. J. Sep. Sci. 2015, 28, 1334–1346.
[CrossRef] [PubMed]
13. Delazar, A.; Nahar, L.; Hamedeyazdan, S.; Sarker, S.D. Microwave-Assisted Extraction in Natural Products Isolation. In Natural
Products Isolation; Sarker, S.D., Nahar, L., Eds.; Humana Press: Totowa, NJ, USA, 2012; pp. 89–115.
14. Gullón, P.; Eibes, G.; Lorenzo, J.M.; Pérez-Rodríguez, N.; Lú-Chau, T.A.; Gullón, B. Green sustainable process to revalorize
purple corn cobs within a biorefinery frame: Co-production of bioactive extracts. Sci. Total Environ. 2020, 709, 136236. [CrossRef]
[PubMed]
15. Zhang, D.Y.; Zu, Y.G.; Fu, Y.J.; Meng, L.; Gu, C.B.; Wei, W.; Yao, X.H. Negative pressure cavitation extraction and antioxidant
activity of biochanin A and genistein from the leaves of Dalbergia odorifera T. Chen. Sep. Purif. Technol. 2011, 83, 91–99. [CrossRef]
16. Gw, A.; Qi, C.A.; Ljy, A.; Ying, L.A.; Mzg, A.; Yao, M.A.; Ji, L.; Sdz, A.; Wei, W.A. Negative pressure cavitation based ultrasound-
assisted extraction of main flavonoids from Flos Sophorae Immaturus and evaluation of its extraction kinetics-ScienceDirect. Sep.
Purif. Technol. 2020, 244, 115805.
17. Kaderides, K.; Papaoikonomou, L.; Serafim, M.; Goula, A.M. Microwave-assisted extraction of phenolics from pomegranate
peels: Optimization, kinetics, and comparison with ultrasounds extraction. Chem. Eng. Process. Process Intensif. 2019, 137, 1–11.
[CrossRef]
18. Da Porto, C.; Porretto, E.; Decorti, D. Comparison of ultrasound-assisted extraction with conventional extraction methods of oil
and polyphenols from grape (Vitis vinifera L.) seeds. Ultrason. Sonochemistry 2013, 20, 1076–1080. [CrossRef]
19. Zhang, H.Y.; Xu, R.Y.; Hao, J.Y.; Wang, Z.X.; Zeng, Y.Q.; Gao, Y. Etection of activity of liquid formula of flavonoid from Lycium
ruthenicum and optimization of extraction proces. Chin. Pharm. J. 2021, 56, 883–890.
20. Sheng, Z.L.; Wan, P.F.; Dong, C.L.; Li, Y.H. Optimization of total flavonoids content extracted from Flos Populi using response
surface methodology. Ind. Crops Prod. 2013, 43, 778–786. [CrossRef]
21. Yin, H.H.; Liao, H.H.; Zhao, W.; Zeng, X.Y.; Tong, Y.M.; Liu, W.; Jiang, Y.M.; Qin, Z.H. Response surface optimization of ultrasonic
assist extraction of triterpenoids from Rhodomyrtus tomentosa leaves and its antioxidant activity. Food Res. Dev. 2021, 42, 74–80.
22. Li, J.; Wu, C.; Li, F.; Yu, R.; Wu, X.; Shen, L.; Liu, Y.; Zeng, W. Optimization of ultrasound-assisted water extraction of flavonoids
from Psidium guajava leaves by response surface analysis. Prep. Biochem. Biotechnol. 2019, 49, 21–29. [CrossRef]
23. Xu, D.P.; Zheng, J.; Zhou, Y.; Li, Y.; Li, S.; Li, H.B. Extraction of natural antioxidants from the Thelephora ganbajun Mushroom by an
ultrasound-assisted extraction technique and evaluation of antiproliferative activity of the extract against human cancer cells. Int.
J. Mol. Sci. 2016, 17, 1664. [CrossRef] [PubMed]
24. Ballesteros, L.F.; Teixeira, J.A.; Mussatto, S.I. Extraction of polysaccharides by autohydrolysis of spent coffee grounds and
evaluation of their antioxidant activity. Carbohydr. Polym. 2017, 157, 258–266. [CrossRef] [PubMed]
25. Li, P.; Zhou, L.; Mou, Y.; Mao, Z. Extraction optimization of polysaccharide from Zanthoxylum bungeanum using RSM and its
antioxidant activity. Int. J. Biol. Macromol. 2015, 72, 19–27. [CrossRef] [PubMed]
26. Duan, M.H.; Luo, M.; Zhao, C.J.; Wang, W.; Zu, Y.G.; Zhang, D.Y.; Yao, X.H.; Fu, Y.J. Ionic liquid-based negative pressure
cavitation-assisted extraction of three main flavonoids from the pigeonpea roots and its pilot-scale application. Sep. Purif. Technol.
2013, 107, 26–36. [CrossRef]
27. Zhou, T.; Luo, C.M.; Zhang, S.L. Dynamic changes of chemical constituents in different drying processes of the leaves from Salvia
miltiorrhiza. Chin. Tradit. Pat. Med. 2019, 41, 2946–2952.
28. Zhou, T.S. Salvianolic acid B in roots of Salvia miltioorrhiza is a dehydration stress induced product during the post-harvested
drying process. Mod. Chin. Med. 2013, 15, 211–218.
29. Dong, J.P. Isolation of Chemical Composition in lxeris sonchifolia (Bge.) Hance and Assaying Collected at Different Time; Jilin Agricultural
University: Jilin, China, 2008.
30. Wang, L.L. Research of dynamic characteristic and dry matter accumulation and active component of Ixeris chinensis. For. Sci.
Technol. 2011, 36, 29–31, 48.
31. Kedare, S.B.; Singh, R.P. Genesis and development of DPPH method of antioxidant assay. J. Food Sci. Technol. 2011, 48, 412–422.
[CrossRef]
32. Zhang, W.M.; Wei, J.; Hu, Z.; Zhang, H.D. Study on extraction and purification of chlorogenic acid from Flos lonicerae flowers and
its antioxidation properties. Food Sci. 2011, 32, 21–25.
Molecules 2023, 28, 3345 15 of 15

33. Huang, X.; Tu, Z.; Xiao, H.; Li, Z.; Zhang, Q.; Wang, H.; Hu, Y.; Zhang, L. Dynamic high pressure microfluidization-assisted
extraction and antioxidant activities of sweet potato (Ipomoea batatas L.) leaves flavonoid. Food Bioprod. Process. 2013, 91, 1–6.
[CrossRef]
34. Chen, X.H.; Chen, G.J.; Wang, Z.R.; Kang, J.Q. A comparison of a polysaccharide extracted from ginger (Zingiber officinale) stems
and leaves using different methods: Preparation, structure characteristics, and biological activities. Int. J. Biol. Macromol. 2020,
151, 635–649. [CrossRef] [PubMed]
35. Wannes, W.A.; Mhamdi, B.; Sriti, J.; Jemia, M.B.; Ouchikh, O.; Hamdaoui, G.; Kchouk, M.E.; Marzouk, B. Antioxidant activities of
the essential oils and methanol extracts from myrtle (Myrtus communis var. italica L.) leaf, stem and flower. Food Chem. Toxicol.
2010, 48, 1362–1370. [CrossRef] [PubMed]

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