separations

Article
Deep Eutectic Solvent-Based Microwave-Assisted Extraction
for the Extraction of Seven Main Flavonoids from
Ribes mandshuricum (Maxim.) Kom. Leaves
Wei Wang 1,2,3,4, *, Si-Qiu Xiao 2,3,4 , Ling-Yu Li 2,3,4 and Qing-Yan Gai 2,3,4, *

1 Institute of Advance Carbon Conversion Technology, Huaqiao University, Xiamen 361021, China
2 College of Chemistry, Chemical Engineering and Resource Utilization, Northeast Forestry University,
Harbin 150040, China
3 Key Laboratory of Forest Plant Ecology, Northeast Forestry University, Harbin 150040, China
4 Engineering Research Center of Forest Bio-Preparation, Northeast Forestry University, Harbin 150040, China
* Correspondence: [email protected] (W.W.); [email protected] (Q.-Y.G.)

Abstract: Flavonoids exhibit many biological properties, so it is very important to find an efficient
and green method to extract them from plant materials. In this paper, DES-MAE (deep eutectic
solvent-based microwave-assisted extraction) technique was developed to extract the seven major
active flavonoids from Ribes mandshuricum leaves, namely, trifolin, isoquercetin, rutin, astragalin,
quercetin, hyperoside, and kaempferol. After the completion of the extraction process, macroporous
adsorption resin was used for the purification of seven flavonoids. The BBD (Box–Behnken design)
method combined with RSM (response surface methodology) was applied to acquire the optimal
operating conditions of DES-MAE. The optimal parameters were: temperature: 54 ◦ C, time: 10 min,
extraction solvent: choline chloride/lactic acid with a 1:2 mass ratio, water content: 25%, and
liquid/solid ratio: 27 mL/g. The yields of the seven target flavonoids were 4.78, 2.57, 1.25, 1.15, 0.34,
0.32, and 0.093 mg/g DW (dry weight), respectively. The direct purification of trifolin, isoquercetin,
rutin, astragalin, quercetin, hyperoside, and kaempferol in DES-MAE solution was achieved by
using macroporous resin X-5. The recoveries were 87.02%, 81.37%, 79.64%, 87.13%, 97.36%, 88.08%,
Citation: Wang, W.; Xiao, S.-Q.; Li, and 99.39%, respectively. The results showed that DES-MAE followed by MRCC (macroporous
L.-Y.; Gai, Q.-Y. Deep Eutectic resin column chromatography) represents a promising approach to extracting and separating active
Solvent-Based Microwave-Assisted components from plants.
Extraction for the Extraction of Seven
Main Flavonoids from Ribes Keywords: DES (deep eutectic solvent); Ribes mandshuricum (Maxim.) Kom.; MAE (microwave-
mandshuricum (Maxim.) Kom. Leaves. assisted extraction); macroporous resin; flavonoids
Separations 2023, 10, 191. https://
doi.org/10.3390/separations10030191

Academic Editor: Wojciech

Piekoszewski 1. Introduction
Received: 9 December 2022 Ribes mandshuricum (Maxim.) Kom. belongs to the Grossulariaceae family, which
Revised: 11 January 2023 belongs to the Saxifragales order. It is also known as Manchurian currant and is one kind
Accepted: 18 January 2023 of deciduous shrub [1]. It is widely distributed in China, Russia, and North Korea and is
Published: 10 March 2023 widely planted in the north of China as a landscape tree. Its berries can traditionally be used
to create jellies, jams, juices, and teas and can also be used as a TCM (traditional Chinese
medicine), known as Denglong-guo or Shan-Yingtao. Its berries contain abundant bioactive
compounds such as flavonoids, phenolic acids, anthocyanins, and vitamins. However, as
Copyright: © 2023 by the authors.
by-products, the leaves have not been fully utilized. A previous chemical study showed that
Licensee MDPI, Basel, Switzerland.
in Ribes mandshuricum leaves, flavonoids were the main bioactive compounds [2]. Because
This article is an open access article
of their multiple biological activities, the major flavonoids such as trifolin, isoquercetin,
distributed under the terms and
rutin, astragalin, quercetin, hyperoside, and kaempferol (Figure 1) attracted more and
conditions of the Creative Commons
more interest. The seven components possess lipid free radical scavenging activity, anti-
Attribution (CC BY) license (https://
viral activity, anti-inflammatory activity, hepatoprotective effects, vasodilatation, analgesic
creativecommons.org/licenses/by/
4.0/).
activity, peroxidation inhibitory activity, antagonistic of PAF (platelet-activating factor),

Separations 2023, 10, 191. https://doi.org/10.3390/separations10030191 https://www.mdpi.com/journal/separations

Separations 2023, 10, x FOR PEER REVIEW 2 of 18

Separations 2023, 10, 191 2 of 18

lipid free radical scavenging activity, anti-viral activity, anti-inflammatory activity,
hepatoprotective effects, vasodilatation, analgesic activity, peroxidation inhibitory activ-
ity, antagonistic of PAF (platelet-activating factor), antidepressant activity, etc. [2–15]. In
antidepressant activity,
order to study and fullyetc. [2–15].
utilize RibesInmandshuricum
order to studyleaves’
and fully Ribes mandshuricum
utilizemedicinal
potential values, we
leaves’ potential medicinal values, we need an efficient method to extract and
need an efficient method to extract and separate them from Ribes mandshuricum leaves. separate
them from Ribes mandshuricum leaves.

Figure 1. Structures of trifolin, isoquercetin, rutin, astragalin, quercetin, hyperoside, and kaempferol
Figure 1. Structures of trifolin, isoquercetin, rutin, astragalin, quercetin, hyperoside, and
in Ribes mandshuricum leaves.
kaempferol in Ribes mandshuricum leaves.
In recent years, “green extraction” has become the focus of attention of the scientific
In recent years,
and industrial “green extraction”
communities has become the
with the strengthening focus ofenvironmental
of human attention of theprotection
scientific
and industrial [16–18].
consciousness communities
With with the strengthening
the development of human
of chemistry environmental
techniques, protection
environmentally
consciousness [16–18]. With the development of chemistry techniques, environmentally
friendly extraction techniques have caught more attention. Choline chloride/urea mixtures
friendly
are extraction
one such techniques
green solvent. haveetcaught
Abbott more
al. (2003) firstattention.
discovered Choline chloride/urea
that choline chloridemix-
and
tures are one such green solvent. Abbott et al. (2003) first discovered that
urea can be used as a solvent liquid through hydrogen bonds, with choline chloride choline chloride
as
andhydrogen
the urea can be usedreceptor
bond as a solvent liquid
and urea asthrough hydrogen
the hydrogen bonds,
bond donor.with choline
Abbott chloride
named the
as the hydrogen
mixtures eutecticbond receptor
mixtures andand
put urea
themasforward
the hydrogen bond donor.
as a concept, openingAbbott named
the way the
for the
application of the eutectic solvent [19]. During the past decade, the application of deep
eutectic solvents has developed in a variety of directions. Recently, the deep eutectic solvent
has been regarded as a promising new green solvent for foodstuffs, cosmetics, and phar-
Separations 2023, 10, 191 3 of 18

maceuticals [16,20]. Studies showed that eutectic solvents can be recycled and recovered
with extremely low toxicities [21]. In general, regular extraction procedures such as soxhlet
extraction, maceration, and heating reflux extraction are conventionally used to extract
natural products from plants. However, the shortcomings of these methods are obvious;
for example, time-consuming and high energy input with unsatisfactory efficiency [22].
Compared with the above methods, microwave-assisted extraction (MAE) has a higher
amount of anti-free radical components and higher yields in plant extracts [23–27]. Thus,
it can be seen that MAE is more effective than other conventional extraction procedures.
Therefore, DES-MAE is a promising method to extract the main flavonoids from Ribes
mandshuricum leaves.
In this research, our goal is to exploit a green and efficient DES-MAE approach to
extract the seven main bioactive flavonoids from Ribes mandshuricum leaves. In addition,
we used macroporous resin, attempting the purification and separation of seven target
flavonoids in extraction [28–31].

2. Materials and Methods

2.1. Plant Materials
Ribes mandshuricum leaves were collected from Heilongjiang Academy of Agricultural
Sciences, P.R. China, on 6 July 2016, which was identified by Professor Bai-Lin Wang from
Heilongjiang Academy of Agricultural Sciences, P.R. China. Then, the air-dried materials
were pulverized by a pulverizer (HX-200A, Yongkang Hardware and Medical Instrument
Plant, Yongkang, China) and thereafter passed through an 80~100 mesh sieve and stored in
an airtight dryer before use.

2.2. Chemicals and Reagents

We purchased choline chloride (>98.0%) from Aladdin Chemistry Co., Ltd. (Shanghai,
China) and bought citric acid (>98.0%), lactic acid (85.0–90.0%), ethylene glycol (>99.5%),
1, 4-butanediol (>99.0%), glucose (>99.0%), and ethanol from Fuchen Chemical Reagents
Co. (Tianjin, China). Trifolin (>98.0%) and rutin (>98.0%) were purchased from Shang-
hai Yuanye Reagent Company (Shanghai, P.R. China). Astragalin (>98.0%), kaempferol
(>98.0%), hyperoside (>98.0%), isoquercetin (>98.0%), and quercetin (>98.0%) were pur-
chased from Nanjing Zelang Biological Technology Co., Ltd. (Jiangsu, China). Phosphoric
acid and acetonitrile were obtained from J & K Chemical Ltd. (Beijing, China). We obtained
deionized water by using a Unique-R20 water-purification system from Research Scientific
Instruments Co. (Xiamen, China). In this study, all solvents were HPLC grade, and all
extracts and standard solutions were filtered through 0.45 µm nylon membranes (Millipore,
Burlington, MA, USA) before use.

2.3. HPLC Analysis

We analyzed the seven flavonoids in samples using an Agilent 1260 series liquid
chromatography system (Agilent, Palo Alto, CA, USA) equipped with a G1316A tempera-
ture column compartment, a G1311C quaternary pump, a G1328C manual injector, and a
G1314F VWD UV detector. Then, we detected the samples with a Phenomenex Curosil-
PFP reverse-phase column (250 × 4.6 mm i.d., 5 µm, Phenomenex, NJ, USA). Gradient
elution was carried out to analyze the samples, solvent A: phosphoric acid/water (0.5:99.5,
v/v) and solvent B: acetonitrile. The gradient conditions were as follows: 10–15% (B),
0–7 min; 15–17% (B), 7–10 min; 17–17% (B), 10–25 min; 17–18% (B), 25–27 min; 18–19% (B),
27–31 min; 19–19% (B), 31–34 min; 19–26% (B), 34–38 min; 26–26% (B), 38–42 min; 26–50%
(B), 42–55 min; 50–10% (B), 55–60 min. The mobile phase flow rate was 0.8 mL/min. Mean-
while, we retained the column temperature at 40 ◦ C, and the injection volume was 20 µL.
Finally, we set the detection wavelength at 360 nm.
This section validated the simultaneous detection of seven major flavonoids in Ribes
mandshuricum leaves. The linearity, sensitivity, precision, and recovery of the method were
tested.
 was 0.8 mL/min. Meanwhile, we retained the column temperature at 40 °C, and the in-
jection volume was 20 μL. Finally, we set the detection wavelength at 360 nm.
This section validated the simultaneous detection of seven major flavonoids in Ribes
mandshuricum leaves. The linearity, sensitivity, precision, and recovery of the method
were tested.
Separations 2023, 10, 191 4 of 18

2.4. Preparation of DESs

In this study, we prepared DESs by heating different compositions of mixtures to
2.4. Preparation of DESs
70–80 °C with stirring in a magnetic stirrer for a certain time until a uniform liquid was
In this
formed. study,
Table we prepared
1 showed the five DESs by heating
different different
DESs systems compositions of mixtures to
prepared.
70–80 ◦ C with stirring in a magnetic stirrer for a certain time until a uniform liquid was
formed. Table 1 showed
Table 1. Different types of the
DESsfive different
(deep eutecticDESs systems
solvents) prepared.
studied.

Abbreviation
Table 1. Different types of DESs HBD HBA
(deep eutectic solvents) studied. Mole Ratio
DES-1 Citric acid Choline chloride 1:1
Abbreviation
DES-2 LacticHBD
acid CholineHBA
chloride Mole Ratio
1:1,1:2,1:3,1:4,1:5
DES-1
DES-3 Citricglycol
Ethylene acid Choline
Choline chloride
chloride 1:1
1:1
DES-2
DES-4 Lactic acid
1,4-butanediol Choline chloride
Choline chloride 1:1,1:2,1:3,1:4,1:5
1:1
DES-3 Ethylene glycol Choline chloride 1:1
DES-5
DES-4 Glucose
1,4-butanediol Choline chloride
Choline chloride 1:1
1:1
DES-5 Glucose Choline chloride 1:1
2.5. Sample Extraction Procedure
2.5.1.
2.5. Microwave
Sample Assisted
Extraction Extraction (MAE)
Procedure
2.5.1. For
Microwave Assisted Extraction
microwave-assisted extraction, (MAE) we used a modified MAS-Ⅱ microwave oven,
which Forwas from Sineo Microwave
microwave-assisted Chemistry
extraction, we usedTechnology
a modified CO.,
MAS-IILtd.microwave
(Shanghai,oven,
P.R.
China).was
which To mount a reflux
from Sineo condenser
Microwave onto the oven,
Chemistry we drilled
Technology CO.,a Ltd.
hole in the outerChina).
(Shanghai, casing,
and
To to mount
mount an agitator
a reflux condenserblade into
onto the
the flask,we
oven, wedrilled
drilleda another holeouter
hole in the in thecasing,
outer casing.
and to
Figure 2 showed a schematic figure of the MAE experimental setup.
mount an agitator blade into the flask, we drilled another hole in the outer casing. Figure 2
showed a schematic figure of the MAE experimental setup.

Figure 2. Schematic figure of experimental setup for MAE.

Figure 2. Schematic figure of experimental setup for MAE.
We added a pulverized leaves sample (1.0 g) to a certain volume of deionized water
We added
and certain a pulverized
volumes leaves
of different sample
kinds (1.0ing)a to
of DESs 100a mL
certain volumeround-bottom
three-neck of deionized water
flask.
and certain volumes of different kinds of DESs in a 100 mL three-neck
Then, we set the flask into the microwave oven and set the microwave radiation power at round-bottom
flask.
600 W.Then,
After we set thetime
a certain flask into the microwave
extraction, we collectedoven and set thecentrifuged
the solutions, microwavethem,radiation
and
power at 600 W. After a certain time extraction, we collected the solutions,
defined the volume with deionized water to 50 mL. We filtered all the samples through a centrifuged
them,
0.45 µmand defined
nylon filter the volume
before withby
detection deionized
HPLC. We water to 50 mL.
executed Weexperiments
all the filtered all the samples
in triplicate.
through a 0.45 μm nylon filter before detection by HPLC. We executed all the experi-
mentsCommon
2.5.2. in triplicate.
Extraction Methods
In order to compare with the MAE method, the following two conventional extraction
methods were adopted. Each sample was analyzed in triplicate.
HRE (heat refluxing extraction): 1.00 g Ribes mandshuricum leaf sample was weighed
precisely, then we accurately put 27 mL DES into a round-bottom flask. Afterwards, we
placed the round-bottom flask into the water bath kettle with a reflux unit at 54 ◦ C for
120 min. We filtered all the samples through a 0.45 µm nylon filter before HPLC detection.
UAE (ultrasound-assisted extraction): 1.00 g Ribes mandshuricum leaf sample was
weighed precisely, then we accurately put 27 mL DES into a round-bottom flask. Thereafter,
we placed the flask into an ultrasonic bath at 54 ◦ C for 40 min. We filtered all the samples
through a 0.45 µm nylon filter before HPLC detection.
Separations 2023, 10, 191 5 of 18

2.6. Experimental Design

In this study, in order to obtain appropriate extraction conditions for the seven major
flavonoids in Ribes mandshuricum leaves, the parameters of DES-MAE were examined sta-
tistically. For the single-factor experiments, 1:1, 1:2, 1:3, 1:4, and 1:5 M ratios of ChCl/lactic
acid (choline chloride/lactic acid) and 10%, 25%, 40%, 55%, and 70% water contents in
ChCl/lactic acid were investigated. Moreover, a 33 factorial portion BBD (Box–Behnken
design) method combined with RSM (response surface methodology) was applied to ac-
quire the optimal combination of the liquid/solid (L/S) ratio (X1 ), temperature (X2 ), and
extraction time (X3 ); the response variable of the seven major flavonoids’ yields is expressed
as Y. To match the model, we employed seventeen tests and three replicates at the center
in the experiment. The average values were matched with a quadratic equation model as
given below:
3 3 3 3
Y = β 0 + ∑ β i Xi + ∑ β ii X2i + ∑ ∑ β ij Xi Xj (1)
i=1 i=1 i=1 j=i+1

where Xi and Xj were the independent variables; Y was the response variable; β0 , βi , βii ,
and βij were the intercept coefficient, linear coefficient, quadratic coefficient, and interaction
coefficient, respectively.
Table 2 showed the coded and actual levels of the variables.

Table 2. Experimental values of Box–Behnken design (BBD) for the extraction of rutin, hyperoside,
isoquercetin, trifolin, astragalin, quercetin, and kaempferol.

Runs Factors Extraction Yield (mg/g DW)

X1 a b X3 c Y1 Y2 Y3 Y4 Y5 Y6 Y7
X2
1 0(25) 0(55) 0(10) 1.250 0.338 2.478 4.696 1.095 0.426 0.110
2 0(25) 1(70) −1(5) 0.931 0.252 1.802 3.554 0.819 0.169 0.037
3 −1(15) −1(40) 0(10) 1.019 0.288 2.047 3.795 0.890 0.032 0.006
4 −1(15) 0(55) −1(5) 0.941 0.254 1.869 3.505 0.822 0.043 0.009
5 1(35) 0(55) 1(15) 1.087 0.237 2.237 4.259 0.996 0.112 0.020
6 −1(15) 0(55) 1(15) 0.978 0.264 1.895 3.575 0.848 0.159 0.022
7 1(35) 0(55) −1(5) 1.222 0.330 2.568 4.826 1.131 0.048 0.007
8 0(25) 1(70) 1(15) 1.062 0.287 1.882 4.028 0.901 0.412 0.092
9 0(25) 0(55) 0(10) 1.197 0.323 2.491 4.761 1.110 0.376 0.095
10 0(25) 0(55) 0(10) 1.280 0.346 2.581 4.631 1.190 0.261 0.128
11 0(25) 0(55) 0(10) 1.282 0.347 2.516 4.561 1.195 0.376 0.109
12 0(25) 0(55) 0(10) 1.243 0.336 2.646 4.963 1.152 0.476 0.113
13 1(35) 1(70) 0(10) 1.103 0.298 2.258 4.344 1.006 0.104 0.031
14 −1(15) 1(70) 0(10) 0.969 0.262 1.875 3.557 0.846 0.263 0.028
15 0(25) −1(40) −1(5) 1.175 0.318 2.377 4.474 1.053 0.030 0.006
16 1(35) −1(40) 0(10) 1.121 0.303 2.326 4.401 1.034 0.026 0.007
17 0(25) −1(40) 1(15) 1.105 0.299 2.263 4.182 0.989 0.028 0.009
a Liquid/solid ratio (mL/g). b Temperature (◦ C). c Extraction time (min).

2.7. Purification and Separation of Seven Major Flavonoids Extracted by DES-MAE

A column (26 mm × 400 mm) wet-packed with 10 g X-5 macroporous resin was used
for the separation and purification of seven major flavonoids extracted by DES-MAE. Briefly,
25 mL of DES-MAE extract solution acquired under the optimum extraction conditions
flowed through the X-5 macroporous resin column at 3 BV/h (bed volume/hour) flow
velocity. We used 210 mL of deionized water to wash the adsorbate-laden column, and then
70% aqueous ethanol (v/v, 250 mL) at 6 BV/h flow velocity to elute. Then, we collected
the elute and detected it by HPLC as mentioned above. Thereafter, we concentrated and
dried the elute by vacuum-rotary evaporation and counted the recoveries of seven target
flavonoids determined by HPLC. We executed all the experiments in triplicate.
 ume/hour) flow velocity. We used 210 mL of deionized water to wash the adsorb-
ate-laden column, and then 70% aqueous ethanol (v/v, 250 mL) at 6 BV/h flow velocity to
elute. Then, we collected the elute and detected it by HPLC as mentioned above. There-
after, we concentrated and dried the elute by vacuum-rotary evaporation and counted
Separations 2023, 10, 191 the recoveries of seven target flavonoids determined by HPLC. We executed all the6 ofex-
18
periments in triplicate.

2.8. Statistical Analysis

2.8. Statistical Analysis
Statistica 8.0 was used to analyze the response surface contour plots of the inde-
Statistica 8.0 was used to analyze the response surface contour plots of the independent
pendent variables, and Origin 9.0 was used to analyze other experimental data statisti-
variables, and Origin 9.0 was used to analyze other experimental data statistically. More-
cally. Moreover, we formulated experimental results in tables and figures as average ±
over, we formulated experimental results in tables and figures as average ± SD (standard
SD (standard deviation).
deviation).

3. Result
3. Result and
and Discussion
Discussion
3.1. HPLC Analysis
The HPLC
HPLC chromatograms
chromatogramsofofthe theseven-target standard
seven-target substance
standard mixture
substance (Figure
mixture 3A)
(Figure
and
3A) DES-MAE
and DES-MAE extraction solutions
extraction (Figure(Figure
solutions 3B) were
3B)shown
were in Figure
shown in3.Figure
This result revealed
3. This result
that all seven
revealed target
that all flavonoids
seven were separated
target flavonoids in both the
were separated standard
in both substances
the standard mixture
substances
and Ribes mandshuricum leaf-extracting solutions.
mixture and Ribes mandshuricum leaf-extracting solutions.

Figure 3. HPLC chromatograms of standard mixture (A) and extracts by DES-MAE in Ribes
Figure 3. HPLC chromatograms of standard mixture (A) and extracts by DES-MAE in Ribes
mandshuricum leaves (B) for (1) Rutin, (2) Hyperoside, (3) Isoquercetin, (4) Trifolin, (5) Astragalin,
mandshuricum leaves (B) for (1) Rutin, (2) Hyperoside, (3) Isoquercetin, (4) Trifolin, (5) Astragalin,
(6) Quercetin, and (7) Kaempferol.
(6) Quercetin, and (7) Kaempferol.
The measuring of the seven flavonoids in Ribes mandshuricum leaves by HPLC was
The measuring of the seven flavonoids in Ribes mandshuricum leaves by HPLC was
validated in this portion. The performance research regulated includes: linearity, sensi-
validated in this portion. The performance research regulated includes: linearity, sensitivity,
tivity, precision, and recovery.
precision, and recovery.
3.1.1. Linearity
3.1.1. Linearity and
and Sensitivity
Sensitivity
The HPLC gradient elutionmethod’s
The HPLC gradient elution method’s linearity
linearity waswas tested
tested based
based on aon a matrix
matrix collo-
collocated
correction model through linear fitting the data at seven concentrations by intercepts inter-
cated correction model through linear fitting the data at seven concentrations by and a
ceptsweighing
1/x and a 1/x weighing
factor. factor. We
We executed executed
the seven theflavonoids’
target seven target flavonoids’
linear linear
regression regres-
analyses by
sion analyses
adopting by adopting
the external the external
standard method.standard method.
The average The replicates
of three average ofwas
three replicates
regarded as
the peak area value. Then, we gathered the data of calibration in Table 3. Each analyte’s
linearity of the standard was well with correlation coefficients being more than 0.995.
Moreover, we appraised the gradient elution method’s sensitivity using the parameters of
limit of detection (LOD) and limit of quantification (LOQ), defined as the target analytes’
minimum concentrations that can be accurately measured and quantified. The LOD and
LOQ were calculated based on a signal-to-noise ratio (SNR) of 3 and 10. Table 3 summarized
the results. The thresholds of LOD and LOQ were extremely low, which showed that the
analytical method was of a very high level of sensitivity.
Separations 2023, 10, 191 7 of 18

Table 3. Regression data and sensitivity for rutin, hyperoside, isoquercetin, trifolin, astragalin,
quercetin, and kaempferol.

Linearity Range LOD b LOQ c

Analyte Calibration Equation a R2
(µg/mL) (µg/mL) (µg/mL)
Rutin 7.813–500 Y = 16364.907X + 114.548 0.20 0.66 0.9952
Hyperoside 7.813–500 Y = 31748.616X + 18.190 0.42 1.42 0.9957
Isoquercetin 7.813–500 Y = 26116.801X + 36.001 0.41 1.37 0.9994
Trifolin 7.813–500 Y = 25721.581X − 11.432 0.19 0.64 0.9996
Astragalin 7.813–500 Y = 30879.312X − 95.355 0.18 0.61 0.9979
Quercetin 7.813–500 Y = 69090.791X − 153.246 0.31 1.03 0.9978
Kaempferol 7.813–500 Y = 82794.569X + 54.854 0.14 0.46 0.9993
a There were 7 different concentrations (n = 3); Y: peak area; X: concentration of standard substance (µg/mL).
b Analyze the concentration of the sample when the signal-to-noise ratio (S/N) was 3. c Analyze the concentration
of the sample when the signal-to-noise ratio (S/N) was 10.

3.1.2. Precision and Recovery

The intra-day and inter-day precisions, that is, the precision of the analytical method,
were evaluated according to RSDs (corresponding relative standard deviations). To deter-
mine the precision of intra-day, we analyzed the samples six times within 24 h, whereas for
the precision of inter-day, we analyzed the samples three times for six consecutive days. The
recovery was determined by using spiked samples of the Ribes mandshuricum leaves matrix.
The results (Table 4) showed that the RSD values of intra-day and inter-day variations for
RT (retention time) were in the range of 0.06–−0.67% and 0.06–−0.62%, respectively. The
RSD values of the intra-day and inter-day variations for the peak area (PA) ranged from
1.81 to 3.41% and 2.45 to 3.13%. The seven target flavonoids’ average recoveries were 97.99
to 99.21% with RSD values less than 4.47% (Table 4). All the above results demonstrated
that the analytical method was appropriate for the determination of trifolin, isoquercetin,
rutin, astragalin, quercetin, hyperoside, and kaempferol in Ribes mandshuricum leaves.

Table 4. Precision and recovery of seven flavonoids (n = 6).

Amount Recovery
Analyte Intra-Day Variations Inter-Day Variations R.S.D. (%)
Added (µg) (%)
RSD for RT RSD for PA RSD for RT RSD for PA
(%) (%) (%) (%)
25 98.97 2.33
Rutin 0.37 2.83 0.36 2.94
50 97.99 3.23
25 98.89 3.18
Hyperoside 0.40 2.55 0.37 2.66
50 98.35 3.66
25 99.21 3.56
Isoquercetin 0.43 2.53 0.40 2.61
50 98.58 2.29
25 99.16 3.10
Trifolin 0.67 3.41 0.62 3.13
50 99.02 3.90
25 98.75 4.47
Astragalin 0.47 2.84 0.44 2.78
50 98.24 3.21
25 99.11 3.78
Quercetin 0.08 1.81 0.07 2.76
50 98.56 3.54
25 98.87 3.16
Kaempferol 0.06 1.87 0.06 2.45
50 98.53 3.68

3.2. Screening of DESs

The different structures of DES determined they have different physicochemical prop-
erties. Consequently, different types of DES had different extraction yields of the seven
target flavonoids. Thus, five different types of ChCl-based DESs were selected. Figure 4A
depicted the extraction yields of the seven flavonoids. Compared with the other two
solvents, using DES-2, DES-4, and DES-5 as solvents obtained higher concentrations of the
 25 98.87 3.16
Kaempferol 0.06 1.87 0.06 2.45
50 98.53 3.68

3.2. Screening of DESs

The different structures of DES determined they have different physicochemical
Separations 2023, 10, 191 properties. Consequently, different types of DES had different extraction yields of the 8 of 18
seven target flavonoids. Thus, five different types of ChCl-based DESs were selected.
Figure 4A depicted the extraction yields of the seven flavonoids. Compared with the
other two solvents,
flavonoids. AlthoughusingtheDES-2,
yield DES-4, and DES-5
of isoquercetin in as
thesolvents obtainedwas
DES-2 solvent higher
much concen-
lower than
trations of the flavonoids. Although the yield of isoquercetin in the DES-2 solvent was
DES-4 and DES-5, the yields of trifolin, quercetin, and kaempferol in the DES-2 solvent
much lower than DES-4 and DES-5, the yields of trifolin, quercetin, and kaempferol in the
were higher than DES-4 and DES-5. The different structures of DESs determined they have
DES-2 solvent were higher than DES-4 and DES-5. The different structures of DESs de-
different physicochemical properties. The hydrogen donor of five DESs was different. The
termined they have different physicochemical properties. The hydrogen donor of five
hydrogen donor of DES-1 and DES-2 was carboxylic acid. The hydrogen donor of DES-3
DESs was different. The hydrogen donor of DES-1 and DES-2 was carboxylic acid. The
and DES-4
hydrogen was of
donor polyalcohol.
DES-3 and DES-4The hydrogen donor ofThe
was polyalcohol. DES-5 was sugar.
hydrogen donor Their
of DES-5viscosity,
polarity,
was acidity
sugar. Their or alkalinity,
viscosity, and ionic
polarity, conductivity
acidity were
or alkalinity, different.
and The viscosity
ionic conductivity wereof DES-1
different. The viscosity of DES-1 and DES-5 were larger than other DESs, the acidity of DESs,
and DES-5 were larger than other DESs, the acidity of DES-2 was stronger than other
and due
DES-2 wasto stronger
the polarity
thanofother
quercetin
DESs,and andkaempferol,
due to theit polarity
was weaker than the and
of quercetin other five
flavonoids. The extraction yields of quercetin and kaempferol by lactic
kaempferol, it was weaker than the other five flavonoids. The extraction yields of quer- acid formed by
DESsand
cetin were higher than
kaempferol by those formed
lactic acid by polyalcohol
formed by DESs were andhigher
sugar.than
In addition,
those formedthe preferable
by
extract yields
polyalcohol andofsugar.
the seven flavonoids
In addition, using DES-2
the preferable as ayields
extract solventof may be due
the seven to their strong
flavonoids
multi-interactions,
using including
DES-2 as a solvent may hydrogen
be due to bonding, π–π,
their strong and ionic/charge-charge
multi-interactions, including withhy- these
flavonoids [32]. After all these comprehensive considerations, DES-2 was chosen as the
drogen bonding, π–π, and ionic/charge-charge with these flavonoids [32]. After all these
comprehensive
extraction solventconsiderations, DES-2 was
for the extraction of thechosen
sevenasmajor
the extraction
flavonoids solvent formandshuricum
in Ribes the ex-
traction
leaves. of the seven major flavonoids in Ribes mandshuricum leaves.

Separations 2023, 10, x FOR PEER REVIEW 9 of 18

Figure 4. Cont.
Separations 2023, 10, 191 9 of 18

Figure 4. Effects of different DESs (A), choline chloride/lactic acid ratio (B), and water content of
DESs (C) on the extraction yield of rutin, hyperoside, isoquercetin, trifolin, astragalin, quercetin, and
Figure 4. Effects of different DESs (A), choline chloride/lactic acid ratio (B), and water content of
kaempferol
DESs in Ribes
(C) on the mandshuricum
extraction leaves.
yield of rutin, hyperoside, isoquercetin, trifolin, astragalin, quercetin,
and kaempferol in Ribes mandshuricum leaves.
3.3. Single-Factor Experiments
3.3.1.
3.3. The EffectExperiments
Single-Factor of ChCl/Lactic Acid Ratio
The Effect
3.3.1. The effect of
of ChCl/Lactic
the ChCl/lactic
Acid acid
Ratioratio was investigated. A range of different ratios of
ChCl/lactic
The effectacid (Figure
of the 4B) was
ChCl/lactic acidexamined for extracting
ratio was investigated. yieldsofofdifferent
A range trifolin,ratios
isoquercetin,
of
rutin, astragalin, quercetin, hyperoside, and kaempferol. The extraction yields
ChCl/lactic acid (Figure 4B) was examined for extracting yields of trifolin, isoquercetin, of the seven
targetastragalin,
rutin, flavonoids were found
quercetin, to increase
hyperoside, and at first and decrease
kaempferol. subsequently.
The extraction With the
yields of the
increase
seven in the
target ratio of were
flavonoids the ChCl/lactic acid rate
found to increase fromand
at first 1:1 decrease
to 1:2 (mol/mol), the seven
subsequently. With target
flavonoids’
the increase inyields reached
the ratio a stable
of the level. acid
ChCl/lactic Therefore,
rate fromfor 1:1
further
to 1:2research,
(mol/mol), wethe
selected
seven DES-2
(ChCl/lactic acid) with a ratio of 1:2 (mol/mol) as the extraction solvent.

3.3.2. The Effect of Water Content of ChCl/Lactic Acid DES

Figure 4C showed the effect of the water content on the flavonoid yields. The seven
target flavonoids were extracted with different water contents from 10% to 70% (v/v) in
ChCl/lactic acid. Rutin and trifolin’s yields reached the highest when the water content was
25% (v/v). The yields of hyperoside, isoquercetin, and astragalin reached the highest when
the content of water was 55% (v/v). The yields of quercetin and kaempferol reached a peak
when the water content was 10% (v/v). In comprehensive consideration, the total yields
of the seven flavonoids were maximal when the water content was 25% (v/v). Therefore,
25% (v/v) water in ChCl/lactic acid was chosen in further experiments to optimize the
parameters of the L/S ratio, extraction time, and temperature.

3.4. Optimization of DES-MAE Using BBD

BBD combined with RSM was used to optimize the L/S ratio, temperature, and extrac-
tion time conditions in DES-MAE. Table 2 showed the experiment results. The mathematical
regression model for the seven major flavonoids is shown below in accordance with the
coded levels:

Ytrifolin = 4.72 + 0.42X1 − 0.17X2 − 0.039X3 + 0.045X1 X2 − 0.16X1 X3 + 0.19X2 X3 − 0.36X1 2 − 0.34X2 2 − 0.32X3 2 (2)

Yisoquercetin = 2.54 + 0.21X1 − 0.15X2 − 0.042X3 + 0.026X1 X2 − 0.089X1 X3 + 0.049X2 X3 − 0.18X1 2 − 0.24X2 2 − 0.22X3 2 (3)
Yrutin = 1.25 + 0.078X1 − 0.044X2 − 4.51 × 10−3 X3 + 8.09 × 10−3 X1 X2 − 0.043X1 X3 + 0.050X2 X3 − 0.10X1 2 − 0.093X2 2 − 0.089X3 2 (4)
Separations 2023, 10, 191 10 of 18

Yastragalin = 1.15 + 0.095X1 − 0.049X2 − 0.012X3 + 3.89 × 10−3 X1 X2 − 0.040X1 X3 + 0.037X2 X3 − 0.098X1 2 − 0.11X2 2 − 0.10X3 2 (5)

Yquercetin = 0.38 − 0.026X1 + 0.10X2 + 0.053X3 − 0.038X1 X2 − 0.013X1 X3 + 0.061X2 X3 − 0.17X1 2 − 0.10X2 2 − 0.12X3 2 (6)
Yhyperoside = 0.34 + 0.012X1 − 0.014X2 − 8.27 × 10−3 X3 + 5.44 × 10−3 X1 X2 − 0.026X1 X3 + 0.014X2 X3 − 0.034X1 2 − 0.016X2 2 − 0.033X3 2 (7)

Ykaempferol = 0.11 + 5.02 × 10−6 X1 + 0.020X2 + 0.011X3 + 4.28 × 10−4 X1 X2 + 1.26 × 10−4 X1 X3 + 0.013X2 X3 − 0.057X1 2 − 0.036X2 2 − 0.039X3 2 (8)
where Y was the target flavonoid’s yield (mg/g DW), X1 was the L/S ratio (mL/g), X2 was
the extraction temperature (◦ C), and X3 was the extraction time (min).
Table 5 showed the ANOVA results in which the determination coefficient (R2 ) values
of the seven major flavonoids were more than 0.90, and the p-values for the seven quadratic
models were significant (p < 0.01), indicating that these quadratic models were suitable for
describing the research response related to the seven target flavonoids. In addition, the
t-test and p-value were used to determine the significance of each coefficient. In all seven
quadratic models, the linear terms of X1 , X2 , and X3 and the quadratic terms of X1 2 and X2 2
were significant (Table 5).
Figure 5 showed the three-dimensional (3D) response surfaces exploited from the
fitted quadratic polynomial equation. In order to express the interactions between the oper-
ating variables and the responses, one independent variable remained at an intermediate
level, whereas other variables changed within the defined ranges. Figure 5A,D,G,J,M,P,S
showed 3D diagrams of the response surface for the yields of trifolin, isoquercetin, rutin,
astragalin, quercetin, hyperoside, and kaempferol as connected with X1 (L/S ratio) and X2
(temperature), respectively. In those plots, it was found that the yields of quercetin and
kaempferol increased with increasing the L/S ratio and extraction temperature, and the
extraction yields of rutin, hyperoside, trifolin, and astragalin increased with increasing
the L/S ratio and decreasing temperature. Raising the L/S ratio from 15 to 32 mL/g
with temperatures from 40 to 65 ◦ C improved the extraction yields of the seven target
flavonoids, but their extraction yields did not continuously improve when the L/S ratio
and temperature exceeded 32 mL/g and 65 ◦ C. As shown in Figure 5B,E,H,K,N,Q,T, the
interaction between the L/S ratio and the extraction time was similar to that between
the L/S ratio and the extraction temperature. The yields of trifolin, isoquercetin, rutin,
astragalin, quercetin, hyperoside, and kaempferol first increased and then decreased with
increasing the L/S ratio and extraction time. Adding the extraction time from 5 to 12 min
improved the extraction yields of the seven flavonoids, whereas their extraction yields did
not continuously improve when the extraction time exceeded 12 min. Figure 5C, F, I, L, O,
R, U showed the extraction yields of rutin, hyperoside, isoquercetin, trifolin, astragalin,
quercetin, and kaempferol as related to X2 (temperature) and X3 (time), respectively. Its
results were similar to that of the above.
In summary, by performing parameter optimization based on the model above, the
optimal conditions for the simultaneous extraction of seven flavonoids from Ribes mand-
shuricum leaves using DES-MAE were: an L/S ratio of 27.34 mL/g, a temperature of
54.08 ◦ C, and a time of 9.79 min. Under these optimal parameters, the predicted extraction
yields of trifolin, isoquercetin, rutin, astragalin, quercetin, hyperoside, and kaempferol
were 4.81, 2.59, 1.27, 1.17, 0.36, 0.34, and 0.11 mg/g DW, respectively. Due to the parameters
being difficult to execute in actual research, an L/S ratio of 27 mL/g, an extraction temper-
ature of 54 ◦ C, and an extraction time of 10 min were selected. In the circumstances, the
predicted extraction yields for trifolin, isoquercetin, rutin, astragalin, quercetin, hyperoside,
and kaempferol were 4.81, 2.59, 1.26, 1.16, 0.35, 0.33, and 0.10 mg/g DW, respectively,
which were approximate to those under optimal conditions. In conclusion, the optimal
conditions for the simultaneous extraction of seven flavonoids from Ribes mandshuricum
leaves using DES-MAE were: an L/S ratio of 27 mL/g, an extraction temperature of 54 ◦ C,
and an extraction time of 10 min.
Separations 2023, 10, 191 11 of 18

Table 5. ANOVA statistics of the model for extraction yields of rutin, hyperoside, isoquercetin, trifolin,
astragalin, quercetin, and kaempferol.

Rutin
Sources
F-Value p-Value
Model 9.69 0.0034 significant
X1 a 20.18 0.0028
X2 b 6.46 0.0385
X3 c 0.067 0.8034
X1 X2 0.11 0.7522
X1 X3 3.06 0.1240
X2 X3 4.18 0.0801
X1 2 18.90 0.0034
X2 2 14.98 0.0061
X3 2 13.73 0.0076
Lack of fit 3.35 0.1365 not significant
R2 0.9257
Hyperoside
Sources
F-value p-value
Model 7.64 0.0069 significant
X1 a 4.69 0.0671
X2 b 5.56 0.0505
X3 c 2.06 0.1946
X1 X2 0.44 0.5262
X1 X3 9.97 0.0160
X2 X3 2.79 0.1387
X1 2 17.93 0.0039
X2 2 4.28 0.0772
X3 2 16.96 0.0045
Lack of fit 5.69 0.0631 not significant
R2 0.9077
Isoquercetin
Sources
F-value p-value
Model 8.74 0.0046 significant
X1 a 22.84 0.0020
X2 b 11.27 0.0121
X3 c 0.91 0.3723
X1 X2 0.17 0.6948
X1 X3 2.00 0.2003
X2 X3 0.60 0.4648
X1 2 8.33 0.0234
X2 2 15.12 0.0060
X3 2 13.17 0.0084
Lack of fit 6.18 0.0555 not significant
R2 0.9183
Separations 2023, 10, 191 12 of 18

Table 5. Cont.

Trifolin
Sources
F-value p-value
Model 10.15 0.0029 significant
X1 a 36.80 0.0005
X2 b 5.96 0.0446
X3 c 0.32 0.5916
X1 X2 0.21 0.6620
X1 X3 2.58 0.1521
X2 X3 3.74 0.0944
X1 2 13.77 0.0075
X2 2 12.40 0.0097
X3 2 11.18 0.0124
Lack of fit 2.54 0.1951 not significant
R2 0.9288
Astragalin
Sources
F-value p-value
Model 9.33 0.0038 significant
X1 a 24.14 0.0017
X2 b 6.49 0.0383
X3 c 0.36 0.5697
X1 X2 0.020 0.8911
X1 X3 2.14 0.1868
X2 X3 1.80 0.2215
X1 2 13.45 0.0080
X2 2 15.92 0.0053
X3 2 14.46 0.0067
Lack of fit 2.06 0.2483 not significant
R2 0.9230
Quercetin
Sources
F-value p-value
Model 9.13 0.0041 significant
X1 a 1.11 0.3269
X2 b 18.05 0.0038
X3 c 4.63 0.0685
X1 X2 1.23 0.3048
X1 X3 0.14 0.7161
X2 X3 3.14 0.1198
X1 2 26.43 0.0013
X2 2 9.50 0.0178
X3 2 12.59 0.0094
Lack of fit 0.42 0.7481 not significant
R2 0.9215
Separations 2023, 10, 191 13 of 18

Separations 2023, 10, x FOR PEER REVIEW 13 of 18

Table 5. Cont.

Kaempferol
temperature,
Sources and the extraction yields of rutin, hyperoside, trifolin, and astragalin in-
F-value p-value
creased with increasing the L/S ratio and decreasing temperature. Raising the L/S ratio
from 15Model
to 32 mL/g with temperatures20.21 from 40 to 65 °C 0.0003 significant
improved the extraction yields of
a
X1 target × 10 -6 0.9992did not continuously improve
the seven flavonoids,1.1
but their extraction yields
b
X2 L/S 17.74 exceeded 32 mL/g 0.0040
when the ratio and temperature and 65 °C. As shown in Figure
X3 c 4.92 0.0621
5B,E,H,K,N,Q,T, the interaction between the L/S ratio and the extraction time was similar
X1 X2 3.992 × 10−3 0.9514
to that Xbetween the L/S ratio and −the extraction temperature. The yields of trifolin,
1 X3 3.453 × 10 4 0.9857
isoquercetin,
X2 X3 rutin, astragalin, quercetin,
3.82 hyperoside, and
0.0914 kaempferol first increased and
then decreased
X1 2 with increasing the L/S ratio and extraction
75.23 <0.0001 time. Adding the extraction
time fromX2 25 to 12 min improved 29.33the extraction yields of the seven flavonoids, whereas
0.0010
X3 2
their extraction yields did not 35.42
continuously improve0.0006 when the extraction time exceeded
Lack
12 min. of fit 5C, F, I, L, O, 1.68
Figure R, U showed the extraction0.3074 yields of rutin,not significant
hyperoside,
R2 trifolin, astragalin,
isoquercetin, 0.9629
quercetin, and kaempferol as related to X2 (temperature)
a Liquid/solid ratio (mL/g). b Temperature (◦ C). c Extraction time (min).
and X3 (time), respectively. Its results were similar to that of the above.

Figure 5. Cont.
Separations 2023, 10, 191 14 of 18
Separations 2023, 10, x FOR PEER REVIEW 14 of 18

Figure
Figure 5. Response
5. Response surfaces
surfaces for extraction
for extraction yields
yields of rutin
of rutin (A–C),(A−C), hyperoside
hyperoside (D–F),(D−F), isoquercetin
isoquercetin (G–I),
(G−I), trifolin (J−L), astragalin (M−O), quercetin (P−R), and kaempferol (S−U) from
trifolin (J–L), astragalin (M–O), quercetin (P–R), and kaempferol (S–U) from Ribes mandshuricumRibes mands-
huricum leaves.
leaves.

In summary,
3.5. Verification of theby performing parameter optimization based on the model above, the
Models
optimal conditions for the simultaneous extraction of seven flavonoids from Ribes
In this paper, in order to validate the reliability of the predicted response values, three
mandshuricum leaves using DES-MAE were: an L/S ratio of 27.34 mL/g, a temperature of
sequential experiments were carried out under optimal conditions. In the circumstances,
54.08 °C, and a time of 9.79 min. Under these optimal parameters, the predicted extrac-
the real extraction yields for trifolin, isoquercetin, rutin, astragalin, quercetin, hyperoside,
Separations 2023, 10, 191 15 of 18

and kaempferol were 4.78, 2.57, 1.25, 1.15, 0.34, 0.32, and 0.093 mg/g DW, respectively,
which were approximate to those predicted values. The RSD values between the predicted
extraction yields of seven target flavonoids and the actual yields were 1.58%, 1.42%, 1.13%,
1.08%, 1.64%, 1.33%, and 1.02%, respectively. This revealed that the optimal extraction con-
ditions obtained were practical and reliable. Therefore, it also showed that the established
quadratic models were rational and reliable.

3.6. Contrast of Conventional Extraction Methods

In order to further prove the yields of the seven target flavonoids by using MAE, a
contrast was performed between the proposed DES-MAE method and the conventional
DES-UAE and DES-HRE methods regarding their performances of trifolin, isoquercetin,
rutin, astragalin, quercetin, hyperoside, and kaempferol extraction from Ribes mandshuricum
leaves (Figure 6). It showed that the extraction yields of trifolin, isoquercetin, rutin, as-
tragalin, quercetin, hyperoside, and kaempferol by the DES-MAE method were much
better than the DES-UAE and DES-HRE methods. The obtained extraction yields of trifolin,
isoquercetin, rutin, astragalin, quercetin, hyperoside, and kaempferol were 4.78, 2.57, 1.25,
1.15, 0.34, 0.32, and 0.093 mg/g DW, respectively, which were higher than those of the
DES-HRE method (3.20, 1.75, 0.85, 0.77, 0.25, 0.014, and 0.0030 mg/g DW, respectively) and
the DES-UAE method (3.91, 2.11, 1.03, 0.87, 0.29, 0.011, and 0.0020 mg/g DW, respectively).
In addition, compared with the conventional extraction methods, DES-MAE takes away
only a small fraction of environmentally friendly extraction solvent. The proposed DES-
MAE approach is a green and effective way for the extraction of trifolin, isoquercetin, rutin,
astragalin, quercetin, hyperoside, and kaempferol from Ribes mandshuricum leaves.

3.7. Purification and Separation of Seven Major Flavonoids from DES-MAE Extraction Solution
In this experiment, the direct separation of trifolin, isoquercetin, rutin, astragalin,
quercetin, hyperoside, and kaempferol from DES solution was executed by making use of
macroporous resin X-5. After one run treatment with X-5 macroporous resin, the contents
of trifolin, isoquercetin, rutin, astragalin, quercetin, hyperoside, and kaempferol reached
2.85%, 1.43%, 0.69%, 0.68%, 0.23%, 0.19%, and 0.06%, respectively. The mass of the extract
was 97.23 mg/g DW. Their contents were 4.30%, 2.16%, 1.04%, 1.03%, 0.34%, 0.30%, and
0.10%, respectively. The recovery yields were 87.02%, 81.37%, 79.64%, 87.13%, 97.36%,
88.08%, and 99.39%, respectively. In a word, X-5 macroporous resin could effectively
purify the seven flavonoids from the extraction solution, whereas the DES solution could
be removed with deionized water, and then the seven target flavonoids could be eluted
with ethanol. In brief, the purification and separation of the seven major flavonoids in the
DES-MAE solution were efficiently and practicably achieved by using X-5 macroporous
resin.
Separations 2023,
Separations 10,10,
2023, x FOR
191 PEER REVIEW 16 of
16 18
of 18

Figure 6. Comparison of different extraction methods on the extraction yields of rutin, hyperoside,
isoquercetin, trifolin, astragalin, quercetin, and kaempferol.
Figure 6. Comparison of different extraction methods on the extraction yields of rutin, hyperoside,
isoquercetin, trifolin, astragalin, quercetin, and kaempferol.
4. Conclusions
In this paper, seven flavonoids were first detected in Ribes mandshuricum leaves. A
3.7. Purification and Separation of Seven Major Flavonoids from DES-MAE Extraction Solution
green and efficient DES-MAE (deep eutectic solvents-based microwave-assisted extraction)
In this experiment,
technique was developed thetodirect
extractseparation
the seven of trifolin,
major activeisoquercetin, rutin, astragalin,
flavonoids trifolin, isoquercetin,
quercetin, hyperoside, and kaempferol from DES solution was executed
rutin, astragalin, quercetin, hyperoside, and kaempferol from Ribes mandshuricum by makingleaves.use
of The
macroporous resin X-5. After one run treatment with X-5 macroporous
ChCl/lactic acid system turned out to be the optimal DES extraction solvent for the resin, the con-
tents of trifolin,
extraction isoquercetin,
of seven rutin, astragalin,
target flavonoids from Ribesquercetin,
mandshuricumhyperoside, and optimal
leaves. The kaempferol yields
reached 2.85%, 1.43%,
were obtained under0.69%, 0.68%, 0.23%,
an extraction 0.19%,ofand
temperature 54 ◦0.06%, respectively.
C, an extraction timeThe mass
of 10 min,ofan
theextraction
extract was 97.23of
solvent mg/g DW. Their
ChCl/lactic acid contents were
with a 1:2 M 4.30%,
ratio, a2.16%, 1.04%, 1.03%,
water content of 25%, 0.34%,
and an
0.30%, and of0.10%,
L/S ratio respectively.
27 mL/g. The recovery
The seven target yields
flavonoids’ yieldswere 87.02%,
reached 81.37%,
4.78, 2.57, 1.25,79.64%,
1.15, 0.34,
87.13%, 97.36%, 88.08%, and 99.39%, respectively. In a word, X-5 macroporous
0.32, and 0.093 mg/g DW, respectively. Based on the experimental results, the DES-MAE resin
could effectively
method obtained purify
highertheyields
sevenandflavonoids from the
took a shorter timeextraction solution, methods
than the common whereas (HREthe
DESandsolution
UAE). could be removed
Meanwhile, it was with
also deionized water,
testified that and isoquercetin,
trifolin, then the seven target
rutin, flavo-
astragalin,
noids could be
quercetin, eluted with
hyperoside, ethanol.
and In brief,
kaempferol in the purification
a DES and separation
extract could be directlyofenriched
the seven and
major flavonoids
separated by X-5inresin.
the DES-MAE
DES-MAEsolution
followedwere efficiently and
by macroporous practicably
resin achieved by
column chromatography
using X-5 macroporous
represents a promising resin.
approach to extracting and separating active compounds from
natural plants for capable application.
4. Conclusions
Author Contributions:
In this paper, seven Supervision;
flavonoidsvalidation,
were firstW.W.; writing—review
detected and editing, S.-Q.X.;
in Ribes mandshuricum writing—
leaves. A
original draft, L.-Y.L.; writing—review and editing, Q.-Y.G. All authors have read and agreed to the
green and efficient DES-MAE (deep eutectic solvents-based microwave-assisted extrac-
published version of the manuscript.
tion) technique was developed to extract the seven major active flavonoids trifolin,
Funding: This
isoquercetin, workastragalin,
rutin, was supported by the National
quercetin, Key R&D
hyperoside, and Program
kaempferolof China
from(2022YFD2200604)
Ribes mands-
and theleaves.
huricum Scientific
TheResearch Fundsacid
ChCl/lactic of Huaqiao
systemUniversity.
turned out to be the optimal DES extraction
solvent for the extraction
Data Availability of seven
Statement: target
All data flavonoids
are contained from
within theRibes mandshuricum leaves. The
article.
optimal yields were obtained under an extraction temperature of 54 °C, an extraction
Conflicts of Interest: The authors declare no conflict of interest.
time of 10 min, an extraction solvent of ChCl/lactic acid with a 1:2 M ratio, a water con-
tent of 25%, and an L/S ratio of 27 mL/g. The seven target flavonoids’ yields reached 4.78,
2.57, 1.25, 1.15, 0.34, 0.32, and 0.093 mg/g DW, respectively. Based on the experimental
results, the DES-MAE method obtained higher yields and took a shorter time than the
common methods (HRE and UAE). Meanwhile, it was also testified that trifolin,
isoquercetin, rutin, astragalin, quercetin, hyperoside, and kaempferol in a DES extract
could be directly enriched and separated by X-5 resin. DES-MAE followed by
Separations 2023, 10, 191 17 of 18

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