Biomass Conversion and Biorefinery

https://doi.org/10.1007/s13399-023-03877-8

ORIGINAL ARTICLE

Efficient extraction, excellent activity, and microencapsulation

of flavonoids from Moringa oleifera leaves extracted by deep eutectic
solvent
Ping Wei1 · Yue Zhang1 · Yao‑Ying Wang1 · Jin‑Feng Dong1 · Bi‑Ni Liao1 · Zhi‑Cheng Su1 · Wu Li1 · Ju‑Cai Xu1 ·
Wen‑Yong Lou2 · Hui‑Hui Su3 · Chao Peng1

Received: 15 October 2022 / Revised: 14 January 2023 / Accepted: 29 January 2023

© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2023

Abstract
A deep eutectic solvent (choline chloride (ChCl)-urea) was chosen to extract flavonoids from Moringa oleifera leaves
(FMOL), the condition of extraction was tailor-made, under the optimal extraction conditions (material-to-liquid ratio of
1:60 g/mL, extraction time of 80 min, extraction temperature of 80 °C), the highest extraction efficiency reached 63.2 ± 0.3 mg
R/g DW, and nine flavonoids were identified. Then, the biological activities including antioxidant activities, antibacterial
activities, and anti-tumor activities were systematically studied. FMOL was superior to positive drugs in terms of antioxi-
dant activity. As to DPPH investigation, the ­IC50 of FMOL and Vc were 64.1 ± 0.7 and 176.1 ± 2.0 µg/mL; for the ABTS,
the ­IC50 of FMOL and Vc were 9.5 ± 0.3 and 38.2 ± 1.2 µg/mL, the FRAP value of FMOL and Vc were 15.5 ± 0.6 and
10.2 ± 0.4 mg TE/g, and ORAC value of FMOL and Vc were 4687.2 ± 102.8 and 3881.6 ± 98.6 µmol TE/g. The bacteriostatic
(MICs were ≤ 1.25 mg/mL) activities of FMOL were much better than propyl p-hydroxybenzoate. Meanwhile, FMOL had
comparable inhibitory activity with genistein on tumor cells, IC50 was 307.8 µg/mL, and could effectively induce apoptosis
in HCT116. Microcapsules were prepared with xylose-modified soybean protein isolate and gelatin as wall materials; after
that, the intestinal release of modified FMOL microcapsules was 86 times of free FMOL. Therefore, this study confirmed
that FMOL extracted with ChCl/urea has rich bioactive components, and microencapsulated FMOL has potential applica-
tion in food industry.

Keywords Flavonoids · Deep eutectic solvent · Moringa oleifera leaves · Identification · Microcapsule

1 Introduction (MOL) also contains polysaccharides, polyphenols, water-

soluble proteins, and different kinds of flavonoids. Extracts
Moringa oleifera is a traditional plant in India, and its flow- of MOL have potential benefits in the antioxidant, antican-
ers, fruits, seeds, leaves, and roots are almost all edible and cer, and hypoglycemic activities [1]. Flavonoids are one of
have high nutritional value [1]. Some studies have reported the main biologically active components of Moringa oleif-
that Moringa oleifera exhibits growth inhibitory activ- era. Various solvents such as water [3], methanol/water [4,
ity against various pathogens [2]. Moringa oleifera leaves 5], methanol [6, 7], ethanol [7, 8], ethanol/water [9, 11],
n-hexane [12, 13], ethyl acetate [6, 12], and chloroform
* Chao Peng fraction [13] combined with assisted extraction methods
[email protected] (homogenizer [4], ultrasound [14, 15], microwave [14] and
1
supercritical fluid extraction [16], etc.) have been used for
School of Biotechnology and Health Sciences, Wuyi extracting flavonoids from Moringa oleifera leaves.
University, Jiangmen 529020, Guangdong, China
2
Traditional organic solvents are the most commonly used
Lab of Applied Biocatalysis, School of Food Science to extract flavonoid compounds. But they are volatile, toxic,
and Engineering, South China University of Technology,
Guangzhou 510640, Guangdong, China highly consumed, which largely lead to excessive solvent
3 residue in the extraction and unfriendly to environment.
Institute of Biological and Medical Engineering, Guangdong
Academy of Sciences, Guangzhou 510316, Guangdong, Deep eutectic solvents (DESs) have emerged at the historical
China moments to address the disadvantage of traditional solvents.

13
Vol.:(0123456789)
 Biomass Conversion and Biorefinery

DESs have been widely used as catalyst, reaction media, were isolated from ChCl/urea. The composition of the
and solvent to extract natural products [17]. As DESs with extracted flavonoids was qualitatively identified. Then, sys-
low raw material price, easy to achieve, low volatility, safe tematically studied the biological activities of the extracted
nontoxicity, convenient to store, and lack of byproducts, flavonoids, including antioxidant activity, antibacterial activ-
they have been exploited to extract bioactive substances ity, and antitumor activity. Moreover, the isolated Moringa
such as flavonoids [18]. Koutsoukos et al. extracted phe- oleifera flavonoids were microencapsulated in order to
nolic and carotenoid compounds from propolis, almond, improve its bioavailability in small intestine.
and red shrimp using DES, prepared by choline chloride/
tartaric acid [19]. Cui et al. prepared 12 kinds of DESs to
extract flavonoids from sea buckthorn leaves [17]. Yu and 2 Materials and methods
Bulone prepared five kinds of choline chloride − based
DESs to extract quercetin derivatives from apple pomace 2.1 Reagents and materials
[20]. Wu et al. attempted to use 13 kinds of DESs, based
choline chloride, betaine, l-proline as hydrogen bond accep- See supplementary materials for materials and reagents.
tors and malic acid, glycerol, oxalic acid, levulinic acid as
hydrogen bond donors with different molar ratios, to extract 2.2 Preparation of the DES
total phenolic and flavonoid of MOL [21]. The extraction
ability of choline chloride/citric acid as DES also has been The DESs list in Table S1 were prepared according to the
demonstrated on dried MOL, two of glucosinolates, eight report [17] with minor modifications. See supplementary
of chlorogenic acids, and twenty of flavonoids have been materials for specific operation.
extracted [22]. Our previous study investigated the extrac-
tion rate of FMOL by choline chloride/lactic acid, and the 2.3 The extraction of FMOL by DES
FMOL can be reached 54.8 mg/g DW, which much higher
than that of extracted by water and ethanol [23]. Different The flavonoids from Moringa oleifera leaves (FMOL) were
DESs have various extraction capacities; therefore, it is valu- extracted by a simple stirring method, and the extraction
able to attemptable to extract FMOL using deep eutectic rate of flavonoids was determined by ultraviolet spectro-
solvent and systematic studies on the biological activity of photometry as Wang’s report [29]. The specific method was
the extracted products. that 0.25 g dry MOL added to 5 mL DES, stirring at 60 °C,
The flavonoids can be broken down into other compounds 200 rpm for 60 min. As shown in Table S1, the solvent with
with lower bioavailability after gastrointestinal digestion, the highest extraction rate of FMOL was selected from the
which may affect by many factors such as pH values, bile six solvents and compared with ethanol and deionized water.
salts, and digestive enzymes in the gastrointestinal tract [24]. Each group of experiments was carried out three times in
Therefore, in addition to optimize the efficient extraction parallel.
of green solvents, it is also necessary to further improve ChCl/urea (1:2, mol/mol) was selected as extraction sol-
the bioavailability of flavonoids. Microencapsulation as a vent; the effects of material-to-liquid ratio (1:10–1:80 g/mL),
versatile technique is commonly used for isolation, pro- extraction time (20–120 min), and temperature (40–90 °C)
tection, and controlled delivery of active core materials on the extraction rate were further studied at the stirring
to enhance the bioavailability and stability of compounds velocity of 200 rpm.
and to improve their controlled release [25, 26]. Soy pro-
tein isolate (SPI) and gelatin (GE) are commonly used as 2.4 Experiment of response surface methodology
wall materials for the preparation of microcapsules. Jia et al.
reported that Maillard reaction modification of whey protein On the basis of single factor experiment, the extraction pro-
and xylooligosaccharide could help to improve the embed- cess was optimized by Box-Behnken design (BBD) with
ding rate and bioavailability [27]. Zhong et al. used a vari- three factors and three levels by using the Design-Expert
ety of oligosaccharides to modify soybean protein isolate 8.0.6 statistical software. The experimental scheme was
through Maillard reaction as the antioxidant wall material shown in Table S2, and Table S3 for factor level table. The
for embedding Lactobacillus casei [28]. In order to improve variables were coded as − 1, 0, and + 1 levels respectively,
the bioavailability of FMOL in intestine, xylose-modified in which the material-to-liquid ratio (X1) were 1:70, 1:80,
soy protein isolate could be used as wall material for micro- and 1:90 g/mL, the extraction duration (X2) were 70, 80,
encapsulation of FMOL. and 90 min, and the extraction temperature (X3) were 70,
In this study, flavonoids were extracted from the leaves 80, and 90 °C. Taking the extraction rate of total flavonoids
of Moringa oleifera using DES (ChCl/urea). The stirring in FMOL (Y1) as the response variable, 17 groups of experi-
extraction conditions were optimized, and the flavonoids ments were designed.

13
Biomass Conversion and Biorefinery

2.5 Detection and identification of FMOL 2.8 Antiproliferative activity assay

components
2.8.1 Antitumor cell activity
An ultra-performance liquid chromatography orbital ion
trap mass spectrometry (UPLC-Q-Orbitrap MS) platform The inhibition rate of tumor cells was determined by the
was used for the qualitative study. See supplementary MTT method. Three types of tumor cells with higher inhi-
materials for specific operation. bition rates were selected from human breast cancer cells
(MCF-7), human colon cancer cells (HCT116), human cer-
vical cancer cells (HeLa), human lung cancer cells (A549),
2.6 Antioxidant activities human liver cancer cells (Hep G-2), and mouse melanoma
cells (B16), and their IC50 were calculated. See supple-
2.6.1 DPPH free radical scavenging activity mentary material for specific operation. Cell inhibition rate
(%) = ­(ODcontrol − ­ODsample)/ODcontrol × 100%. All tests were
The DPPH radical scavenging rate of the sample was performed in sextuplicate.
determined as previous report [23]. See supplementary Acridine orange (AO) staining experiment: 1 mL/well
material for specific operation. cell suspension (5 × ­105 cells/mL) was added into each cell
culture plate, and cultured in 5% ­CO2 at 37 °C for 24 h.
Then each well was added 1 mL FMOL sample (diluted
2.6.2 ABTS free radical scavenging activity
with culture medium) and further incubated for 3 h. After
the incubation, each sample was stained with 20 µL AO flu-
The clearance rate of ­ABTS+ free radicals was determined
orescent dye solution (5 µg/mL) for 0.5 h, and then washed
according to the previous report [23]. See supplementary
with PBS for 3 times, after that 1 mL of culture medium
material for specific operation.
was added to each well. Then, the samples were observed
under a fluorescence inverted microscope or laser confocal
2.6.3 Iron reducing antioxidant capacity (FRAP) microscope.

FRAP capability of FMOL was determined by previous report 2.8.2 The effect of FMOL on apoptosis
[23]. See supplementary material for specific operation.
The apoptosis experiment was determined according to the
reported method with slightly modified [31]. See supple-
2.6.4 Oxygen Free Radical Absorption Capacity (ORAC) mentary material for specific operation.

The oxygen radical absorbance capacity (ORAC) was 2.9 Preparation of microcapsules

evaluated by comparison with Trolox using the method
[30]. See supplementary material for specific operation. Xylose (xyl) was selected to modify soybean protein isolate
(SPI) by Maillard reaction [27], and FMOL microcapsules
were prepared by combining gelatin (GE) as crosslinking
2.7 Antimicrobial activity agent with modified SPI by composite coagulation method
[28]. Microcapsule embedding rate (EE) = A − A0/A × 100,
The inhibition zone was determined by the filter paper where A is the content of FMOL in microcapsules, A ­ 0 is
agar diffusion method. Added 300 µL of test bacteria sus- the surface flavonoids attachment of FMOL microcapsules.
pension (1.5 × ­106 CFU/mL) to agar medium, took 6 mm The factors of microcapsule preparation included explor-
diameter sterile filter paper with sterile forceps and stuck ing the effects of different wall material concentration,
it, dropped 10 µL of sample to be tested, cultured upside core-wall ratio, emulsifying power, and emulsifying time
down in 37 °C incubator for 24 h, and measured and on microcapsule embedding rate and morphology. The spe-
counted the diameter of a bacteriostatic circle. cific was shown in supplementary material.
The minimum inhibitory concentration (MIC) has been
determined. 5 µL sample was mixed with 195 µL of test 2.10 Characterization of FMOL microcapsules
bacterial suspension (1.5 × ­106 CFU/mL) and incubated
at 37 °C for 24 h. The OD was measured at 610 nm with The characterization methods of microcapsules were shown
multifunctional microplate reader. The positive control in supplementary material, include Fourier transform infra-
was propyl hydroxybenzoate, and the negative control was red spectroscopy (FT-IR), X-ray diffraction (XRD) [32], and
DMSO. All tests were performed in triplicate. scanning electron microscope (SEM) [33].

13
 Biomass Conversion and Biorefinery

2.11 In vitro digestion of FMOL microcapsules 1:60 and 1:80 g/mL; the rates have little difference, namely
50.7 ± 0.9 and 51.7 ± 0.2 mg R/g DW, respectively, showing
The simulated in vitro digestion of FMOL microcapsules that the extraction approached to platform stage, so the best
was investigated and the general procedure was followed material-to-liquid ratio was regarded as 1:60 g/mL. Such a
Brodkorb’s study [34]. The simulated oral digestive juice, result could contribute to that when the material-to-liquid
gastric digestive juice, and small intestinal digestive juice ratio was too low, the MOL powder was not fully dispersed
were shown in Table S4 and preheated at 37 °C before use. and mixed insufficiently with the DES, leading to an insuffi-
The digestive stabilities of FMOL or FMOL microcapsules cient dissolution of flavonoids. However, an excessively high
in different digestive stages, that is, bioavailability, were material-to-liquid ratio would cause the solvent to absorb a
studied by simulating the digestion of oral, gastric, and intes- certain amount of heat and increase the viscosity, which is
tinal fluids. The release rate of flavonoids (%) = C/C0 × 100, averse to the breaking of MOL cell’s wall, decreasing mass
where C was the content of flavonoids in the solution after transfer, and results in low extraction rate of flavonoids. In
in vitro digestion, and C0 is the content of flavonoids in the the meantime, it could also cause solvent waste and prob-
coating extract before in vitro digestion. lems of subsequent test treatment [36]. Therefore, MOL
powder’s optimal material-to-liquid ratio to ChCl/urea sol-
2.12 Statistical analysis vent was considered to be 1:60 g/mL.
Under the material-to-liquid ratio of 1:60 g/mL, the effect
The data were processed with Origin 2019, and the final of extraction time on the extraction rate of flavonoids was
results were expressed as the mean ± standard deviation of studied. As Fig. 1(c) depicts, with extraction time increas-
three values (n = 3). The Design-Expert 8.0.6 (STAT ease ing from 20 to 120 min, the extraction rate of flavonoids
Inc., Minneapolis, Minnesota, USA) was used for response increased from 29.4 ± 0.04 to 56.1 ± 1.3 mg R/g DW. When
surface methodology (RSM) as an optimization tool. The the extraction time reached 80 min, the highest extraction
IC50 results were statistically analyzed using GraphPad rate (60.6 ± 0.9 mg R/g DW) was obtained, but when the
prism version 8.0 (GraphPad Software Inc., La Jolla, CA), extraction time was increased continuously to 120 min, the
and the significance level was 5%. extraction rate decreased to 56.1 ± 1.3 mg R/g DW. This
could be allied to the extraction time being too long and
thus the flavonoids being destroyed.
3 Results and discussions Then, the optimal extraction temperature of MOL pow-
der extracted with ChCl/urea was further investigated
3.1 The screening of DES under the as-obtained optimum material-to-liquid ratio and
extraction time. As shown in Fig. 1(d), as the temperature
Six deep eutectic solvents were selected as extraction sol- improved from 20 to 80 °C, the extraction rate increased
vents. Figure 1(a) compares the extraction rate of flavo- from 27.1 ± 0.3 to 61.7 ± 0.8 mg R/g DW. In comparison,
noids between six DESs (composed of choline chloride and when the temperature was increased continuously to 90 °C,
various hydrogen bond donors) and traditional solvents. the extraction rate descended to 53.6 ± 2.1 mg R/g DW. This
The extraction rates of FMOL conducted with DESs were may be owing to when the extraction temperature was too
all higher than those obtained with ethanol and DI water. low, the molecular movement was slow and led to a low
DESs composed of diverse hydrogen bond donors and cho- dissolution, diffusion, and penetration rate, which goes
line chloride had different extraction effects on FMOL. This against to the dissolution of flavonoids. High temperature
could be attributed to the different physical and chemical could reduce the viscosity of DES, increase the diffusion
properties of various DESs, including solubility, viscosity, coefficient and improve the extraction efficiency. However,
and polarity, which could affect the extraction efficiency of high temperature could lead to the structure damage of fla-
FMOL [35]. The DES prepared by ChCl/urea had the highest vonoids, resulting in a reduction in the extraction rate.
rate for FMOL extraction in the investigated solvents. The variance analysis was conducted on the test data, and
the bias regression coefficients of each factor were tested.
3.2 The effects of different conditions on the FMOL The analysis of variance results is summarized in Table S5.
extraction rate The larger the F value, the greater the influence. It could
be seen that the order of factors affecting the content of fla-
The control variable method was used to study three sin- vonoids was B (extraction time) > C (extraction tempera-
gle factors. As Fig. 1(b) shows, with the material-to-liquid ture) > A (material-to-liquid ratio).
ratio of 1:10 to 1:80 g/mL, the extraction rate of FMOL The P value of this model was < 0.01, extremely significant,
increased from 34.0 ± 0.5 to 51.7 ± 0.2 mg R/g DW, while and the P′ value of lack of fit was 0.0967 > 0.01, not signifi-
the extraction rates of the material-to-liquid ratio were cant, indicating that the regression equation fit well and could

13
Biomass Conversion and Biorefinery

Fig. 1  a Screening and com- (a) 50 (b) 70

parison of extraction solvents.

Flavonoids extraction rate (mg/g dry-MOL)

Flavonoids extraction rate (mg/g dry-MOL)

Extraction rate of flavonoids 38.0 60
40
using ChCl/urea under different 50.7 51.7
50
b material-to-liquid ratios, c 31.0
28.0
29.5 28.6
45.2
30 26.5
extraction times, and d extrac- 24.3 40 38.0
34.0
tion temperatures. Response
surface diagram of interaction, 20 30
e A: material-to-liquid ratio 20
and B: extraction time, f A: 10
9.1

material-to-liquid ratio and C: 10

extraction temperature, g B: 0
extraction time and C: extrac- 0

O
ly

er
1:10 1:20 1:40 1:60 1:80

no
re
l/C
l/E

l/P

D
G

at
tion temperature, h comparison

ha
l/U

l/B
l/T

Iw
hC
hC

hC
Material-to-liquid ratio (g/mL)

Et
hC

hC
hC

D
C
C

C
of extraction rate before and

C
after optimization (c) 70 C (d) 70

Flavonoids extraction rate (mg/g dry-MOL)

61.7
Flavonoids extraction rate (mg/g dry-MOL)

60.6 59.1 61.0

60 56.1 60
53.6
51.7 51.7
50 50

40 40
33.9
30 29.4 30 28.8
27.1

20 20

10 10

0 0
20 40 60 80 100 120 40 50 60 70 80 90
Extracting time (min) Extraction temperature (oC)

(h) 70
63.2
Flavonoids extraction rate (mg/g dry-MOL)

61.7
60

50

40 38.0

30
24.3
20

10

0
Ethanol Before Single After
optimization factor optimization

predict the content of flavonoids under different extraction con- To further investigate the interaction of relevant variables
ditions. The correlation coefficient R2 of the correction model and determine the best advantage, a response surface graph
was 0.9804, and the correction determination coefficient Radj2 was drawn using the Design-Expert 8.0.6 software for vis-
was 0.9551, indicating that the fitting of the equation was good. ual analysis. The results were consistent with the results of
The model could explain the change of 98.04% response value. mathematical model analysis in Fig. 1(e)–(g), which could
Therefore, this model could analyze the real changes in response better understand the interaction between the two variables
values and determine the optimal extraction process conditions. and its optimal range [35].

13
 Biomass Conversion and Biorefinery

Model verification: The optimal extraction conditions, cynaroside, quercetin 3-β-d glucoside, kaempferol, luteo-
including material-to-liquid ratio, extraction time, and lin, and taxifolin have been reported in Moringa oleifera
temperature, were 1:79 g/mL, 70 min, and 83 °C. Under leaves extracted by hot water [22], ethanol [9], methanol or
these conditions, the predicted flavonoid content in MOL methanol/water [30, 39], choline chloride/lactic acid [23],
was 63.3 ± 0.1 mg R/g DW. The experimental results 20% ammonium sulfate and ethanol [22], choline chloride/
showed that the measure value of total flavonoids in MOL citric acid [22], l-proline/levulinic [21], and l-proline/glyc-
was 63.2 ± 0.3 mg R/g DW, and the error was only 0.1 mg erol [21], and fisetin has been reported in Moringa oleifera
R/g DW. Furthermore, it can be seen from Fig. 1(h) that leaves for the first time.
the extraction rate of flavonoids after optimization was Further analysis of the extracted products obtained with
63.2 ± 0.3 mg mg R/g DW, which was 1.7 and 2.6 times different solvents revealed various flavonoids. Table S7
to that of before optimization (38.0 ± 0.9 mg R/g DW) shows part of the reported results of extracted flavonoids,
and ethanol extraction (24.3 ± 0.8 mg R/g DW). The opti- indicated that the DES is a good solvent for the extraction
mized value was significantly higher than the screening of natural products. Different DESs could sometimes obtain
value (38.0 ± 0.9 mg R/g DW), indicating that the extrac- various extracted products, and new compounds from Mor-
tion conditions of FMOL were successfully optimized inga oleifera leaves.
via this model. Additionally, the optimal extraction rate
of FMOL (63.2 ± 0.3 mg R/g DW) extracted by ChCl/ 3.4 Bioactivity of FMOL
urea from Moringa oleifera leaves was much higher than
that of those extracted with other solvents, such as the 3.4.1 Antioxidant activities
extract yields extracted with 50% aqueous ethanol solution
(29.5 ± 0.3 mg/g DW) [11], methanol (35.19 mg/g DW) [37],
l-proline/ethylene glycol (30.93 mg GAE/g DW) [38], and DPPH free radical scavenging activity of FMOL Flavonoids
l-proline/glycerol (29.8 mg GAE/g DW) [21]. from Moringa oleifera have a significant antioxidant capac-
ity related to its immune enhancement activity [40], anti-
3.3 Identification of FMOL inflammatory activities [41], antioxidant activities [23], and
anticancer activities [1]. Previous studies showed that the
The composition of flavonoids extracted with ChCl/urea was determination of antioxidant activity depended on the reaction
identified using LC–MS, the results were presented in Fig. 2 mechanism, and a variety of tests must be combined to accu-
and Table S6. Nine of flavonoids were identified, includ- rately assess the antioxidant capacity of samples [42]. In this
ing hyperoside, vitexin, quercetin, cynaroside, quercetin study, DPPH, ABTS, FRAP, and ORAC methods were used
3-β-d glucoside, kaempferol, taxifolin, luteolin, and fise- to assess the antioxidant activity of FMOL samples reliably.
tin; the MS diagram of the nine flavonoids could be seen The DPPH radical scavenging ability of the samples was
from Fig. S5–S13, in which hyperoside, vitexin, quercetin, determined. As Fig. 3(a) depicts, it was gratifying that the

8
(a) 7
6
5
Intensity/107

5
4
6,7
6
3
2 8
1 2,3,4
Intensity/107

0
8.60 8.65 8.70 8.75 8.80
Time/min

4 1
6
9
Intensity/107

5
4
3

2
2
1
8.12 8.16 8.20 8.24
Time/min

0
0 2 4 6 8 10 12
Time/min

Fig. 2  a LC–MS chromatogram of FMOL extract, b molecular structure formula of flavonoids

13
Biomass Conversion and Biorefinery

FMOL extracted with ChCl/urea showed excellent DPPH ORAC value of FMOL (4687.2 ± 102.8 µmol TE/g), which
radical scavenging activity, superior to Vc in the range of undoubtedly demonstrated that the flavonoids extracted by
12.5–800 µg/mL. The scavenging rate was 81.4% ± 1.1 DES have superior antioxidant activity than those extracted
at 800 µg/mL. The IC50 of Vc and FMOL values were by the traditional solvent.
170.6 ± 2.0 and 64.1 ± 0.7 µg/mL, respectively. FMOL Through the above comprehensive evaluation of the
samples’ DPPH free radical scavenging activity was more antioxidant capacity of FMOL by the DPPH, ABTS, FRAP,
than twice as high as Vc. The satisfactory DPPH scavenging and ORAC, the excellent antioxidant capacity of FMOL
activity of FMOL was found to be far better than those of extracted via ChCl/urea was demonstrated better than Vc.
many flavonoid extracts from plants, such as Moringa oleif-
era leaf extract (149.8 μg/mL) [43], and hawthorn samples
(80 µg/mL and 120 µg/mL) [30]. 3.4.2 Antimicrobial activity

ABTS free radical scavenging activity of FMOL ABTS free With the abuse of antibiotics, plants have attracted consid-
radical solution had high absorption at 734 nm when FMOL erable attention as a potential source of antibacterial drugs
was added to ABTS free radical solution, the absorbance at [40]. Bacterial growth inhibition is one of methods to study
734 nm decreased, that is, ABTS free radicals were removed antimicrobial activity assessment, which is low cost and
by FMOL, indicating that FMOL had the activity of scaveng- can provide meaningful effect within a short period of time.
ing free radicals [44]. As Fig. 3(b) shows, the scavenging Therefore, it was of great significance to explore the inhibi-
activity of FMOL extracted with ChCl/urea was much bet- tory effect of FMOL on different bacteria.
ter than that of Vc at low concentrations (12.5 ~ 50 µg/mL). The experimental results in Table 2 and Fig. S1 show that
In contrast, the scavenging activity of FMOL was equal to the diameter of the inhibition zone produced by FMOL at
that of Vc at high concentrations (100 ~ 400 µg/mL). FMOL a concentration of 2.5 mg/mL (15.6 ± 0.4–18.7 ± 0.6 mm)
exhibits higher activity in scavenging free radicals even at was even more significant than that of the positive drug at
low concentrations, further demonstrating the excellent anti- 10 mg/mL (13.6 ± 0.4–15.2 ± 1.3 mm), which indicates that
oxidant capacity of the as-obtained FMOL. In the meantime, the inhibitory effects of FMOL on five bacteria were better
the IC50 of FMOL was 9.5 ± 0.3 µg/mL, which was much than that of propyl p-hydroxybenzoate (the positive control).
better than Vc (38.2 ± 1.2 µg/mL). The inhibitory effect on Proteus common was showing best
among the investigated bacteria.
Ferric reducing antioxidant power (FRAP) of FMOL It is To further explore the antimicrobial capacity of FMOL
important to estimate the total antioxidant activity by extracted with ChCl/urea, the minimum inhibitory con-
diverse methods for the total antioxidant potential of any centration (MIC) was investigated by the above five bac-
food matrix, so the FRAP of FMOL was also studied. teria. As shown in Table 2, the MICs of FMOL to E. coli,
The FRAP of FMOL extracted with ChCl/urea was meas- Staphylococcus aureus, Bacillus subtilis, and Pseudomonas
ured, the value obtained was 15.5 ± 0.6 mg TE/g (Table 1), aeruginosa were 1.25 mg/mL and to Proteus common was
and the corresponding value of the positive control Vc was 0.625 mg/mL. The MIC values of the positive drug (propyl
10.2 ± 0.4 mg TE/g. Rocchetti et al. investigated the FRAP p-hydroxybenzoate) were twice to that of FMOL, further
of flavonoid samples from Moringa oleifera leaves extracted revealing the strong bacteriostatic ability of the as-prepared
by ethyl acetate, and the corresponding value was 13.77 mg FMOL. In addition, the MIC results were consistent with the
TE/g [4]. The FRAP of FMOL extracted with ChCl/urea inhibitory zone experiment results.
(15.5 ± 0.6 mg TE/g) was not only higher than that of Vc
(10.2 ± 0.4 mg TE/g) but also superior to that of flavonoids 3.4.3 Antiproliferative activity
extracted via ethyl acetate (13.77 mg TE/g), indicating that
the DES-extracted FMOL has outstanding ferric reducing Epidemiological studies have shown that various parts of
antioxidant capacity. Moringa oleifera have beneficial effects on health, and the
leaves are potential source of antitumor activity [39]. Bio-
Oxygen Free Radical Absorption Capacity (ORAC) of logical evaluation of FMOL was performed with an in vitro
FMOL The ORAC was measured and the results was shown antitumor MTT assay via six types of tumor cells (MCF-7,
in Table 1; the value was 4687.2 ± 102.8 µmol TE/g, supe- HCT116, HeLa, A549, B16, Hep G-2).
rior to that of the positive control Vc (3881.6 ± 98.6 µmol According to the MTT experimental results in Fig. 3(c),
TE/g). Recently, the ORAC values of flavonoids extracted FMOL showed relatively high inhibitory activity against
from Moringa oleifera leaves using water /methanol (20:80, B16, HCT116, and Hep G-2 cells. Different concentrations
v/v) and water/ ethanol (20:80, v/v) were 108.1 µmol TE/g of FMOL were used to the three kinds of cells to obtain dif-
and 3560.5 µmol TE/g [45], respectively, far below the ferent cell inhibitory activities, as shown in Fig. 3(d)–(f).

13
 Biomass Conversion and Biorefinery

13
 Biomass Conversion and Biorefinery

◂Fig. 3  a DPPH radical scavenging activities of Vc and FMOL; b the as-extracted FMOL; furthermore, HCT116 cells exhib-
ABTS radical scavenging activities of Vc and FMOL. c Inhibition ited increasing detached and dead cells with the increasing
of FMOL on various tumor cells, d inhibitory activity of FMOL/
genistein at different concentrations on B16 cells, e inhibitory activ-
dosage of FMOL, indicating the excellent antiproliferative
ity of FMOL/genistein at different concentrations on HCT116 cells, activity of FMOL extracted with ChCl/urea.
f inhibitory activity of FMOL/genistein at different concentrations on In order to further demonstrate the bioactivity of FMOL
Hep G-2 cells on the tumor cells, the effect of FMOL on cellular apop-
tosis was investigated. HCT116 cells were treated with
The IC50 was for the inhibition rates of the three kinds of FMOL at varying concentrations for 24 h by employing
cells. The results displayed that the IC50 of FMOL on B16, an Annexin V-FITC/PI assay. As shown in Fig. 4(e)–(j),
HCT116, and Hep G-2 cells were 415.0 ± 3.3, 307.8 ± 5.8, incubation with FMOL and genistein drastically increased
and 349.6 ± 8.1 µg/mL, respectively. The IC50 of genistein the proportion of apoptotic cells compared with the control
(the positive control) on B16, HCT116, and Hep G-2 cells group. After treatment with FMOL, the proportions of early
were 398.2 ± 10.1, 281.7 ± 4.2, and 335.8 ± 6.7 µg/mL, apoptotic cells were 1.8% at 100 µg/mL, 5.1% at 500 µg/
respectively. Based on the results of the IC50 of antitumor mL, and 6.0% at 1000 µg/mL, respectively, whereas the
proliferation in the three kinds of cells, FMOL had the corresponding proportions of late apoptotic cells reached
best inhibitory effect on HCT116 cells, and it was slightly 4.6%, 9.4%, and 11.2%, respectively. These results illus-
lower than that of genistein. Recently, Yang et al. revealed trated that FMOL significantly induced cellular apopto-
that peanut extract has a strong antiproliferative effect on sis in a dose-dependent manner and thus could effectively
HCT116 cells, with IC50 of 1.39 ~ 9.33 mg dry extract/ induce apoptosis in HCT116 cells.
mL [46], far higher than that of as-extracted FMOL, which
indicated that FMOL has a considerable inhibitory effect 3.5 Characterization of FMOL microcapsules
on tumor cells.
An acridine orange (AO) staining experiment has been The preparation process is shown in Fig. 5(a). As depicts in
performed for FMOL to verify the results from the MTT assay. Fig. S14, the optimal wall material concentration, core-wall
The overlap effect of AO staining results for B16 (Fig. S2), ratio, and stirring rate and time were of 1.8% SPI-3.6% xyl
HCT116 (Fig. S3), and Hep G-2 cells (Fig. S4) became more and 1.0% GE, 1:6, 500 rpm, and 15 min, under which con-
evident with increasing FMOL concentration, and its variation ditions, the embedding rate of FMOL could reach 66.5%,
effect was consistent with the IC50 results. This result and the FMOL microcapsule particle size distribution was
indicated that the pH value in cells increased under the shown in Fig. S15, 94% of the FMOL microcapsule diameter
action of FMOL, and it was speculated that FMOL had a simi- were < 100 µm, observing the morphology of FMOL microcap-
lar effect on alkaline lysosomes [47]. Figure 4 (a)–(d) shows sule through microscope and SEM. The microscope diagram
confocal laser imaging of HCT116 cells stained with AO after of wet microcapsule is shown in Fig. 5(b), and the SEM results
3 h of treatment with FMOL. The overlapping effect of AO are shown in Fig. 5(c)–(d). The morphology of microcapsules
staining revealed dose-dependent effect on cell viability for showed a certain bonding block, there was no obvious parti-
cle dispersion state, and only a few cystic protrusions could
Table 1  FRAP and ORAC values of FMOL and Vc be seen in the sample. The reason for this phenomenon may
Sample FRAP (mg TE/g) ORAC (µmol TE/g) be that some residual wall material polymers did not form
microcapsules during the preparation of microcapsules, or the
FMOL 15.5 ± 0.6 4687.2 ± 102.8 water loss during the freeze drying process after the prepara-
Vc 10.2 ± 0.4 3881.6 ± 98.6 tion of microcapsules may cause the adhesion on the surface

Table 2  Inhibitory results of FMOL against five strains

Strains DMSO Propyl p-hydroxybenzoate FMOL
Diameter of inhibition MIC Diameter of inhibition MIC Diameter of inhibition MIC
zone (D/mm) (mg/mL) zone (D/mm) (mg/mL) zone (D/mm) (mg/mL)

E. coli — — 13.6 ± 0.4 2.5 16.6 ± 0.2 1.25

Staphylococcus aureus — — 15.2 ± 1.3 2.5 16.3 ± 0.7 1.25
Bacillus subtilis — — 14.4 ± 1.4 2.5 17.3 ± 0.5 1.25
Proteus vulgaris — — 14.8 ± 1.1 1.25 18.7 ± 0.6 0.625
Pseudomonas aeruginosa — — 14.2 ± 0.8 2.5 15.6 ± 0.4 1.25

13
 Biomass Conversion and Biorefinery

13
 Biomass Conversion and Biorefinery

◂Fig. 4  Confocal laser imaging of HCT116 cells stained with acridine The bioavailability of free FMOL and microencapsu-
orange after 3 h of a cells without sample treatment, b–d the cells lated FMOL have been investigated; the release amounts
treated with different concentrations of FMOL were extracted by
ChCl/urea: b 50 µg/mL, c 100 µg/mL, d 200 µg/mL. Under differ-
of FMOL, unmodified FMOL microcapsules, and modified
ent treatments: e cells without sample treatment, f–h the cells treated FMOL microcapsules in different simulated digestion stages
with different concentrations of FMOL: f 100 µg/mL, g 500 µg/mL, h were further analyzed. Figure 5(g) depicts that the release
1000 µg/mL, i Genistein (1000 µg/mL), induced HCT116 cell apop- amounts of free FMOL, unmodified FMOL microcapsules,
tosis for 24 h, and j the proportions of apoptosis cells
and modified FMOL microcapsules in simulated oral diges-
tion stages were 57.2%, 26.3%, and 24.0%, respectively. In
of film-forming wall materials, resulting in agglomeration and the simulated gastric digestion stage, the release amounts
shrinkage of microcapsules [28]. were 29.9%, 31.9%, and 33.0%, respectively. In the simulated
Then, the FMOL microcapsules were characterized. It can intestinal digestion stage, the release rates were 0.5%, 32.8%,
be seen from Fig. 5(e) that the characteristic absorption of and 39.4%, respectively. The experimental results showed
amide C = O stretching and amide II bands (N–H and C–N) that the oral release of FMOL was twice of microcapsules,
in protein was around 1650 and 1540 ­cm−1, and the char- which was not conducive to the embedding protection of fla-
acteristic absorption of carbohydrates (including C–C and vonoids. There was little difference in the release of the three
C–O stretching vibration and C–H bending) was mainly con- samples in the simulated gastric digestion stage. The release
centrated at 1180–953 ­cm−1 [28]. Herein, the peak enhance- of unmodified FMOL microcapsules was 49.0 ± 0.5 μg/mL;
ment of wave number 1650 ­cm−1 and the peak attenuation of the release rate was 32.8%, which was 70 times to that of free
1540 ­cm−1 probably be the result of glycosylation between FMOL. In the simulated intestinal digestion stage, the release
SPI and xylose. FMOL microcapsules contained the char- of free FMOL was only 0.7 ± 0.3 μg/mL; the release rate was
acteristic peaks of SPI, xyl, GE, and FMOL in the range only 0.5%. The release amount of modified FMOL microcap-
of 500–4000 ­cm−1, and two characteristic peaks of FMOL sule was 60.2 ± 0.6 μg/mL; the release rate was 39.4%, which
(about 3440 ­cm−1 and 2950 ­cm−1) could be found in the greatly increased the intestinal release of free FMOL (about
infrared spectrum of FMOL microcapsules. Therefore, it 86 times). The intestinal release rate of modified FMOL
could be seen that the wall material had been wrapped into microcapsules was about 6.6% higher than that of unmodi-
FMOL to form FMOL microcapsules. fied FMOL microcapsules. Therefore, the microencapsulation
In general, crystalline materials exhibit spikes, while of FMOL was conducive to protect the slow release of FMOL
amorphous products exhibit wider peaks. As shown in Fig. 5 in the process of digestion, and the microcapsule prepared
(f), in the XRD patterns of all materials, a characteristic by soybean protein isolate modified by xylose could further
diffraction peak appears at about 20°. However, xylose, as a retard the release of FMOL in the intestine.
crystalline substance, showed many peaks between 10 and
80°, but the characteristic peaks of xylose after maillard
reaction did not appear in the characteristic peaks of FMOL 4 Conclusion
microcapsules. The reason may be that xylose could carry
out maillard reaction efficiently at 80 °C and 30 min, which Different DESs were investigated to extract FMOL, and
completely changed its structure [32]. ChCl/urea was found to be the best DES for obtaining
FMOL, with an optimized extractive effect (63.2 ± 0.3 mg
3.6 In vitro digestion of FMOL microcapsules R/g DW). Nine flavonoids were identified by LC–MS and
luteolin was previously unreported from Moringa oleifera
Microcapsules have been widely used in the field of food, leaves. Then, its antioxidant, antibacterial, and antitumor
and many of microencapsulation of food bioactive sub- activities were systematically studied. FMOL was superior
stances are to release active ingredients in the digestive sys- to positive drugs in antioxidant activity (DPPH free radical
tem in a way of effective digestion and absorption, and give scavenging, ABTS free radical scavenging, FRAP, ORAC)
play to their physiological and health effects. and antibacterial activity in vitro. The FMOL extracted
Table 3 shows that the flavonoid release and bioavail- with ChCl/urea had exciting antineoplastic activity and was
ability of free FMOL, modified FMOL microcapsules, and comparable with that of the positive control. The FMOL
unmodified FMOL microcapsules in the simulated oral, has certain antitumor activity, and the overlap effect of AO
gastric, and intestinal stages. The results showed that the staining and cell apoptosis results revealed a dose-depend-
bioavailability of modified FMOL microcapsules increased ent effect on cell viability for the as-extracted FMOL. The
9.1% and unmodified FMOL microcapsules increased 2.4% microencapsulation has enhanced the slow release rate of
compared with the free FMOL. Therefore, the application FMOL, the release amounts of FMOL, unmodified FMOL
of modified microcapsules of FMOL could improve the bio- microcapsules, and modified FMOL microcapsules were
availability of Moringa flavonoids. 0.7 ± 0.3, 60.2 ± 0.6, and 49.0 ± 0.5 µg/mL respectively.

13
 Biomass Conversion and Biorefinery

(e) Modified microcapsule

1540
(g) 60 57.2 FMOL
1650
free FMOL Unmodified microcapsule
Transmittance (a.u.)

Modified microcapsule
Unmodified microcapsule
Flavonoid release rate/%

GE 50
free FMOL SPI 39.4
40
GE Modified microcapsule 33.0 32.8
29.9 31.9
Xyl Unmodified microcapsule 30 26.3
24.0
20
1650 1540 SPI
10
Xyl
0.5
4000 3500 3000 2500 2000 1500 1000 500 10 20 30 40 50 60 0
Wavenumber (cm-1) Oral Stomach Intestinal

Fig. 5  a Microcapsule preparation flow chart. b microcapsule micrograph. SEM images of microcapsules: c 100 μm, d 10 μm. e Infrared charac-
terization of microcapsules. f XRD characterization of microcapsules. g Simulating the release rate of flavonoids during digestion

13
Biomass Conversion and Biorefinery

Table 3  Content and Samples Free FMOL Unmodified Modified

bioavailability of total (μg/mL) microcapsule (μg/mL) microcapsule
flavonoids in different digestion Digestive stage
(μg/mL)
stages
Initial concentration 133.9 ± 1.8 149.5 ± 1.3 152.7 ± 1.3
Oral 76.6 ± 1.6 39.4 ± 0.5 36.7 ± 1.0
Stomach 116.6 ± 2.2 87.1 ± 0.8 87.1 ± 0.9
Intestine 117.3 ± 1.3 136.1 ± 0.3 147.3 ± 1.5
Intestinal release (µg/mL) 0.7 ± 0.3 49.0 ± 0.5 60.2 ± 0.6
Biological accessibility/% 87.6 91.0 96.5

The intestinal release of unmodified FMOL microcapsules 2. Alzaabi MM, Hamdy R, Ashmawy NS, Hamoda AM, Alkhayat F,
and modified FMOL microcapsules were 70 and 86 times Khademi NN, . . . Soliman SSM (2022) Flavonoids are promising
safe therapy against COVID-19. Phytochem Rev 21(1): 291–312.
of that of free FMOL. Therefore, the microencapsulation https://​doi.​org/​10.​1007/​s11101-​021-​09759-z
can improve the bioavailability of FMOL. In conclusion, 3. Olvera Aguirre G, Mendoza Taco MM, Moo Huchin VM, Lee
FMOL extracted by ChCl/urea has excellent biological Rangel HA, Roque Jiménez JA, Gómez Vázquez A, . . . Chay
activity, and its microencapsulation is helpful to FMOL Canul AJ (2022) Effect of extraction type on bioactive compounds
and antioxidant activity of Moringa oleifera Lam. leaves. Agricul-
slower release, which could broad the application pros- ture 12(9): 1462. https://​doi.​org/​10.​3390/​agric​ultur​e1209​1462
pects of FMOL in food or pharmaceutical industry. 4. Rocchetti G, Pagnossa JP, Blasi F, Cossignani L, Piccoli RH,
Zengin G, . . . Lucini L (2020) Phenolic profiling and in vitro
Supplementary Information The online version contains supplemen- bioactivity of Moringa oleifera leaves as affected by different
tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 13399-0​ 23-0​ 3877-8. extraction solvents. Food Res Int 127: 108712. https://​doi.​org/​10.​
1016/j.​foodr​es.​2019.​108712
Author contribution Ping Wei: conceptualization, investigation, for- 5. Aldossary HM, Alqurashi RM (2021) In vitro antioxidant and
mal analysis, writing—original draft, writing—editing. Yue Zhang: antimicrobial activity of Moringa oleifera leaf as a natural food
methodology, investigation, data treatment, formal analysis. Yao-Ying preservative in chicken burgers. Emir J Food Agr 33(6):450–457.
Wang: methodology, software, investigation, writing—original draft. https://​doi.​org/​10.​9755/​ejfa.​2021.​v33.​i6.​2711
Jin-Feng Dong: formal analysis, validation. Bi-Ni Liao: writing—edit- 6. Adeyi AO, Mustapha KK, Ajisebiola BS, Adeyi OE, Metibemu
ing. Zhi-Cheng Su: formal analysis. Wu Li: writing—editing. Ju-Cai DS, Okonji RE (2021) Inhibition of Echis ocellatus venom metal-
Xu: writing—editing. Wen-Yong Lou: writing—editing. Hui-Hui Su: loprotease by flavonoid-rich ethyl acetate sub-fraction of Moringa
writing—editing. Chao Peng: project administration, funding acquisi- oleifera (Lam.) leaves: in vitro and in silico approaches. Toxin Rev
tion, supervision, writing—review. 41(2): 476–486. https://​doi.​org/​10.​1080/​15569​543.​2021.​18933​34
7. Bennour N, Mighri H, Eljani H, Zammouri T, Akrout A (2020)
Funding This work was supported by Guangdong Basic and Effect of solvent evaporation method on phenolic compounds and
Applied Basic Research Foundation (No. 2020A1515110427, the antioxidant activity of Moringa oleifera cultivated in Southern
2022A1515010009, 2020A1515110406), the Youth Innovation Pro- Tunisia. S Afr J Bot 129:181–190. https://​doi.​org/​10.​1016/j.​sajb.​
ject of Guangdong Province University (No. 2020KQNCX088), the 2019.​05.​005
National Natural Science Foundation of China (No. 21802019), Sci- 8. Albarri R, Şahin S (2022) A green method for the extraction of
ence and Technology Program of Jiangmen City (JZ202210), Graduate Moringa oleifera leaves: evaluation of several in vitro assays for
Education Innovation Program of Wuyi University (YJS-SF JD-22–04), bioactive properties. Biomass Convers Bioref. https://​doi.​org/​10.​
the Characteristics Innovation Project of Guangdong Province Uni- 1007/​s13399-​022-​02690-z
versity (No. 2019KTSCX185), COVID-19 Epidemic Prevention and 9. Chigurupati S, Al-Murikhy A, Almahmoud SA, Almoshari Y,
Control Project (No. 2020FKZX02), and the Science and Technology Saber Ahmed A, Vijayabalan S, . . . Raj Palanimuthu V (2022)
Program of Jiangmen (No. 2020030101110005138). Molecular docking of phenolic compounds and screening of anti-
oxidant and antidiabetic potential of Moringa oleifera ethanolic
Declarations leaves extract from Qassim region, Saudi Arabia. Saudi J Biol Sci
29(2): 854–859. https://​doi.​org/​10.​1016/j.​sjbs.​2021.​10.​021
Ethical approval With no need for ethical approval. 10. Silva LMP, Inacio MRC, Silva G, Silva J, Luz J, Almeida MDG, . .
. Zucolotto SM (2022) The first optimization process from cultiva-
Competing interests The authors declare no competing interests. tion to flavonoid-rich extract from Moringa oleifera Lam. leaves in
Brazil. Foods 11(10): 1452. https://​doi.​org/​10.​3390/​foods​11101​452
11. Kashaninejad M, Blanco B, Benito Román O, Beltrán S, Niknam
SM, Sanz MT (2021) Maximizing the freeze-dried extract yield
References by considering the solvent retention index: extraction kinetics and
characterization of Moringa oleifera leaves extracts. Food Bioprod
Process 130:132–142. https://​doi.​org/​10.​1016/j.​fbp.​2021.​09.​008
1. Dhakad AK, Ikram M, Sharma S, Khan S, Pandey VV, Singh 12. Haroen U, Syafwan, Kurniawan K, Budiansyah A (2022) Determi-
A (2019) Biological, nutritional, and therapeutic significance of nation of nutrient content, beta-carotene, and antioxidant activity
Moringa oleifera Lam. Phytother Res 33(11):2870–2903. https://​ of Moringa oleifera extraction using organic solution. J Adv Vet
doi.​org/​10.​1002/​ptr.​6475 Anim Res 9(2):246–254. https://​doi.​org/​10.​5455/​javar.​2022.​i590

13
 Biomass Conversion and Biorefinery

13. Do BH, Hoang NS, Nguyen TPT, Ho NQC, Le TL, Doan CC 26. Lopez Polo J, Monasterio A, Cantero Lopez P, Osorio FA (2021)
(2021) Phenolic extraction of Moringa Oleifera leaves induces Combining edible coatings technology and nanoencapsulation
caspase-dependent and caspase-independent apoptosis through for food application: a brief review with an emphasis on nanoli-
the generation of reactive oxygen species and the activation of posomes. Food Res Int 145:110402. https://​doi.​org/​10.​1016/j.​
intrinsic mitochondrial pathway in human melanoma cells. Nutr foodr​es.​2021.​110402
Cancer 73(5):869–888. https://​doi.​org/​10.​1080/​01635​581.​2020.​ 27. Jia C, Cao D, Ji S, Lin W, Zhang X, Muhoza B (2020) Whey
17768​85 protein isolate conjugated with xylo-oligosaccharides via mail-
14. Albarri R, Şahin S (2022) Monitoring the recovery of bioactive lard reaction: characterization, antioxidant capacity, and appli-
molecules from Moringa oleifera leaves: microwave treatment vs cation for lycopene microencapsulation. LWT-Food Sci Technol
ultrasound treatment. Biomass Convers Bioref. https://​doi.​org/​10.​ 118:108837. https://​doi.​org/​10.​1016/j.​lwt.​2019.​108837
1007/​s13399-​021-​02232-z 28. Zhong S-R, Li M-F, Zhang Z-H, Zong M-H, Wu X-L, Lou W-Y
15. Yerena Prieto BJ, Gonzalez Gonzalez M, Vázquez Espinosa M, (2021) Novel antioxidative wall materials for Lactobacillus casei
González Peredo AV, García Alvarado MÁ, Palma M, . . . Bar- microencapsulation via the maillard reaction between the soy pro-
bero, G. F., (2022). Optimization of an ultrasound-assisted extrac- tein isolate and prebiotic oligosaccharides. J Agric Food Chem
tion method applied to the extraction of flavonoids from Moringa 69(46):13744–13753. https://​doi.​org/​10.​1021/​acs.​jafc.​1c029​07
Leaves (Moringa oleífera Lam.). Agron 12(2): 261–273. https://​ 29. Wang Y, Duan X, Ren G, Liu Y (2019) Comparative study on
doi.​org/​10.​3390/​agron​omy12​020261 the flavonoids extraction rate and antioxidant activity of onions
16. Yerena Prieto BJ, Gonzalez Gonzalez M, García Alvarado MÁ, treated by three different drying methods. Dry Technol 37(2):245–
Casas L, Palma M, del Carmen Rodríguez Jimenes G, . . . Cejudo 252. https://​doi.​org/​10.​1080/​07373​937.​2018.​14829​07
Bastante C (2022) Evaluation of the effect of different co-solvent 30. Li M, Chen X, Deng J, Ouyang D, Wang D, Liang Y, . . . Sun Y
mixtures on the supercritical ­CO2 extraction of the phenolic com- (2020) Effect of thermal processing on free and bound phenolic
pounds present in Moringa oleifera Lam. Leaves. Agron 12(6): compounds and antioxidant activities of hawthorn. Food Chem
1450-1462. https://​doi.​org/​10.​3390/​agron​omy12​061450 332: 127429. https://​doi.​org/​10.​1016/j.​foodc​hem.​2020.​127429
17. Cui Q, Liu JZ, Wang LT, Kang YF, Meng Y, Jiao J, Fu YJ (2018) 31. Wu CJ, Wu JQ, Hu Y, Pu S, Lin Y, Zeng Z, . . . Chen WH (2021)
Sustainable deep eutectic solvents preparation and their efficiency Design, synthesis and biological evaluation of indole-based 1,2,4
in extraction and enrichment of main bioactive flavonoids from triazolo 4,3-a pyridine derivatives as novel microtubule polymeri-
Sea buckthorn leaves. J Clean Prod 184:826–835. https://​doi.​org/​ zation inhibitors. Eur J Med Chem 223: 113629. https://​doi.​org/​
10.​1016/j.​jclep​ro.​2018.​02.​295 10.​1016/j.​ejmech.​2021.​113629
18. Duan L, Dou LL, Guo L, Li P, Liu EH (2016) Comprehensive 32. Meng FB, Li JJ, Zhang Q, Li YC, Liu DY, Chen WJ, Zhang Y
evaluation of deep eutectic solvents in extraction of bioactive nat- (2021) Complex wall materials of polysaccharide and protein
ural products. ACS Sustain Chem Eng 4(4):2405–2411. https://​ effectively protected numb-taste substance degradation of Zanth-
doi.​org/​10.​1021/​acssu​schem​eng.​6b000​91 oxylum bungeanum. J Sci Food Agri 101(11):4605–4612. https://​
19. Koutsoukos S, Tsiaka T, Tzani A, Zoumpoulakis P, Detsi A (2019) doi.​org/​10.​1002/​jsfa.​11103
Choline chloride and tartaric acid, a natural deep eutectic solvent 33. Shao P, Xuan S, Wu W, Qu L (2019) Encapsulation efficiency and
for the efficient extraction of phenolic and carotenoid compounds. controlled release of Ganoderma lucidum polysaccharide micro-
J Clean Prod 241:118384. https://​doi.​org/​10.​1016/j.​jclep​ro.​2019.​ capsules by spray drying using different combinations of wall
118384 materials. Int J Biol Macromol 125:962–969. https://​doi.​org/​10.​
20. Yu L, Bulone V (2021) De-glycosylation and enhanced bioactiv- 1016/j.​ijbio​mac.​2018.​12.​153
ity of flavonoids from apple pomace during extraction with deep 34. Brodkorb A, Egger L, Alminger M, Alvito P, Assuncao R, Bal-
eutectic solvents. Green Chem 23(18):7199–7209. https://d​ oi.o​ rg/​ lance S, . . . Recio I (2019) INFOGEST static in vitro simulation
10.​1039/​d1gc0​1928g of gastrointestinal food digestion. Nat Protoc 14(4): 991–1014.
21. Wu L, Li L, Chen S, Wang L, Lin X (2020) Deep eutectic solvent- https://​doi.​org/​10.​1038/​s41596-​018-​0119-1
based ultrasonic-assisted extraction of phenolic compounds from 35. Mansur AR, Song NE, Jang HW, Lim TG, Yoo M, Nam TG (2019)
Moringa oleifera L. leaves: optimization, comparison and antioxi- Optimizing the ultrasound-assisted deep eutectic solvent extraction
dant activity. Sep Purif Technol 247:117014. https://​doi.​org/​10.​ of flavonoids in common buckwheat sprouts. Food Chem 293:438–
1016/j.​seppur.​2020.​117014 445. https://​doi.​org/​10.​1016/j.​foodc​hem.​2019.​05.​003
22. Hamany Djande CY, Piater LA, Steenkamp PA, Madala NE, 36. Rajha HN, Darra NE, Hobaika Z, Boussetta N, Vorobiev E,
Dubery IA (2018) Differential extraction of phytochemicals from Maroun RG, Louka N (2014) Extraction of total phenolic com-
the multipurpose tree, Moringa oleifera, using green extraction pounds, flavonoids, anthocyanins and tannins from grape byprod-
solvents. S Afr J of Bot 115:81–89. https://​doi.​org/​10.​1016/j.​sajb.​ ucts by response surface methodology. Influence of solid-liquid
2018.​01.​009 ratio, particle size, time, temperature and solvent mixtures on the
23. Wang YY, Peng C, Zhang Y, Wang ZR, Chen YM, Dong JF, . . . optimization process. Pol J Food Nutr Sci 05(04):397–409. https://​
Wei P (2022) Optimization, identification and bioactivity of fla- doi.​org/​10.​4236/​fns.​2014.​54048
vonoids extracted from Moringa oleifera leaves by deep eutectic 37. Rocchetti G, Blasi F, Montesano D, Ghisoni S, Marcotullio MC,
solvent. Food Biosci 47: 101687. https://​doi.​org/​10.​1016/j.​fbio.​ Sabatini S, . . . Lucini L (2019) Impact of conventional/non-con-
2022.​101687 ventional extraction methods on the untargeted phenolic profile
24. Peanparkdee M, Borompichaichartkul C, Iwamoto S (2021) Bio- of Moringa oleifera leaves. Food Res Int 115: 319–327. https://​
accessibility and antioxidant activity of phenolic acids, flavonoids, doi.​org/​10.​1016/j.​foodr​es.​2018.​11.​046
and anthocyanins of encapsulated Thai rice bran extracts during 38. Zhu H, Zhang J, Li C, Liu S, Wang L (2020) Morinda citrifolia L.
in vitro gastrointestinal digestion. Food Chem 361:130161. https://​ leaves extracts obtained by traditional and eco-friendly extraction
doi.​org/​10.​1016/j.​foodc​hem.​2021.​130161 solvents: relation between phenolic compositions and biological
25. George TT, Oyenihi AB, Rautenbach F, Obilana AO (2021) Char- properties by multivariate analysis. Ind Crop Prod 153. https://d​ oi.​
acterization of Moringa oleifera leaf powder extract encapsulated org/​10.​1016/j.​indcr​op.​2020.​112586
in maltodextrin and/or gum arabic coatings. Foods 10(12):3044– 39. Coppin JP, Xu Y, Chen H, Pan M.-H, Ho C.-T, Juliani R, . . .
3062. https://​doi.​org/​10.​3390/​foods​10123​044 Wu Q (2013) Determination of flavonoids by LC/MS and

13
Biomass Conversion and Biorefinery

anti-inflammatory activity in Moringa oleifera. J Func Foods 5(4): as affected by induced ripening agents. Food Chem 339:127909.
1892–1899. https://​doi.​org/​10.​1016/j.​jff.​2013.​09.​010 https://​doi.​org/​10.​1016/j.​foodc​hem.​2020.​127909
40. Li C, Dong Z, Zhang B, Huang Q, Liu G, Fu X (2020) Structural 45. Oldoni TLC, Merlin N, Bicas TC, Prasniewski A, Carpes ST, Ascari
characterization and immune enhancement activity of a novel J, . . . Thome G (2021) Antihyperglycemic activity of crude extract
polysaccharide from Moringa oleifera leaves. Carbohyd Polym and isolation of phenolic compounds with antioxidant activity from
234:115897. https://​doi.​org/​10.​1016/j.​carbp​ol.​2020.​115897 Moringa oleifera Lam. leaves grown in Southern Brazil. Food Res
41. Lizbeth Martinez-Gonzalez C, Martinez L, Martinez Ortiz EJ, Int 141: 110082. https://​doi.​org/​10.​1016/j.​foodr​es.​2020.​110082
Eva Gonzalez Trujano M, Deciga Campos M, Ventura Martinez 46. Yang QQ, Kim G, Farha AK, Luo Q, Corke H (2020) Phenolic
R, Diaz Reval I (2017) Moringa oleifera, a species with potential profile, antioxidant and antiproliferative activities of diverse pea-
analgesic and anti-inflammatory activities. Biomed Pharmacother nut cultivars. J Food Meas Charact 14(5):2361–2369. https://​doi.​
87:482–488. https://​doi.​org/​10.​1016/j.​biopha.​2016.​12.​107 org/​10.​1007/​s11694-​020-​00483-4
42. Zhao DR, Jiang YS, Sun JY, Li HH, Luo XL, Zhao MM (2019) 47. Yu XH, Hong XQ, Mao QC, Chen WH (2019) Biological effects
Anti-inflammatory mechanism Involved in 4-ethylguaiacol-medi- and activity optimization of small-molecule, drug-like synthetic
ated inhibition of LPS-induced inflammation in THP-1 cells. J anion transporters. Eur J Med Chem 184:111782. https://​doi.​org/​
Agric Food Chem 67(4):1230–1243. https://​doi.​org/​10.​1021/​acs.​ 10.​1016/j.​ejmech.​2019.​111782
jafc.​8b062​63
43. Guillén Román CJ, Guevara González RG, Rocha- Guzmán NE, Publisher's note Springer Nature remains neutral with regard to
Mercado-Luna A, Pérez Pérez MCI (2018) Effect of nitrogen pri- jurisdictional claims in published maps and institutional affiliations.
vation on the phenolics contents, antioxidant and antibacterial
activities in Moringa oleifera leaves. Ind Crop Prod 114:45–51. Springer Nature or its licensor (e.g. a society or other partner) holds
https://​doi.​org/​10.​1016/j.​indcr​op.​2018.​01.​048 exclusive rights to this article under a publishing agreement with the
44. Maduwanthi SDT, Marapana R (2021) Total phenolics, flavo- author(s) or other rightsholder(s); author self-archiving of the accepted
noids and antioxidant activity following simulated gastro-intes- manuscript version of this article is solely governed by the terms of
tinal digestion and dialysis of banana (Musa acuminata, AAB) such publishing agreement and applicable law.

13