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Article
Dandelion (Taraxacum mongolicum) Extract Alleviated
H2O2-Induced Oxidative Damage: The Underlying Mechanism
Revealed by Metabolomics and Lipidomics
Yannan Chen 1,2 , Siyuan Fei 1,2 , Xiaoting Yu 1,2 and Mingqian Tan 1,2, *

1 Academy of Food Interdisciplinary Science, School of Food Science and Technology, Dalian Polytechnic
University, Qinggongyuan, Gangjingzi District, Dalian 116034, China; ynchen04@163.com (Y.C.);
13470319363@163.com (S.F.); xiaoting12ming@126.com (X.Y.)
2 National Engineering Research Center of Seafood, Dalian Polytechnic University, Dalian 116034, China
* Correspondence: mqtan@dlpu.edu.cn; Tel.: +86-411-86318657

Abstract: Dandelion has received wide attention in food and medicine fields due to its excellent
antioxidant properties. Nonetheless, the underlying mechanism of this action has not yet been
fully clarified, particularly at the metabolic level. Herein, the effects of dandelion extract (DE) on
H2 O2 -induced oxidative damage was investigated. The results indicate that the DE alleviated H2 O2 -
induced cell damage (increased by 14.5% compared to H2 O2 group), reduced the reactive oxygen
species (ROS) level (decreased by 80.1% compared to H2 O2 group), maintained the mitochondrial
membrane potential (MMP) level, and increased antioxidant-related enzyme activities. Importantly,
the metabolic response of PC12 cells indicates that H2 O2 disturbed phospholipid metabolism and
damaged cell membrane integrity. In addition, energy metabolism, the central nervous system, and
the antioxidant-related metabolism pathway were perturbed. In contrast, DE rescued the H2 O2 -
induced metabolic disorder and further alleviated oxidative damage. Collectively, these findings
provide valuable stepping stones for a discussion of the mechanism and show the promise of DE as a
suitable additive for functional food products.

Keywords: dandelion; antioxidant; oxidative damage; metabolomics; lipidomics

Citation: Chen, Y.; Fei, S.; Yu, X.; Tan,
M. Dandelion (Taraxacum mongolicum)
Extract Alleviated H2 O2 -Induced
Oxidative Damage: The Underlying
1. Introduction
Mechanism Revealed by
Metabolomics and Lipidomics. Foods Oxidative stress is considered as one of the key factors related to human patholog-
2023, 12, 3314. https://doi.org/ ical processes, such as diabetes, chronic inflammation, cancers, and neurodegenerative
10.3390/foods12173314 diseases [1,2]. The abundance of reactive oxygen species (ROS) under oxidative stress leads
to damage to cellular macromolecules, including protein modification, lipid oxidation, and
Academic Editor: Kuiwu Wang
DNA damage, which further causes the derangement of cellular functions and accelerates
Received: 10 August 2023 the occurrence of diseases [3]. In contrast, it is well known that a diet rich in antioxidants,
Revised: 28 August 2023 especially the natural antioxidants, has a positive effect on maintaining the balance of ROS
Accepted: 29 August 2023 concentration and preventing a variety of pathophysiological processes [4]. Moreover,
Published: 3 September 2023 treatment based on supplements containing natural antioxidants lacks many of the side
effects related to pharmacological treatment.
Dandelion (Taraxacum mongolicum), a member of the Asteraceae family, is widely used
in traditional Chinese medicine and food supplements owing to its specific biological
Copyright: © 2023 by the authors.
activities, such as anti-bacterial, anti-neoplastic, anti-inflammatory, and hepatoprotective
Licensee MDPI, Basel, Switzerland.
properties, especially its antioxidant effect [5–7]. The different components from different
This article is an open access article
distributed under the terms and
organs of dandelion have demonstrated a strong anti-oxidative action [8,9]. For example,
conditions of the Creative Commons
Jedrejek et al. found the phenolic fractions from the leaves of dandelion showed antioxidant
Attribution (CC BY) license (https:// activity against H2 O2 -induced oxidative damage [10]. Sun et al. confirmed dandelion
creativecommons.org/licenses/by/ aqueous extract could effectively alleviate LPS-induced oxidative stress in MAC-T cells [11].
4.0/). Additionally, dandelion polysaccharides also showed significant antioxidant activity in

Foods 2023, 12, 3314. https://doi.org/10.3390/foods12173314 https://www.mdpi.com/journal/foods

Foods 2023, 12, 3314 2 of 14

various scavenging models [12]. Nevertheless, the current study of the effect of dandelion
extract against oxidative damage is primarily focused on experimental investigations; the
underlying mechanism of actions is not yet clear and needs to be discussed in depth.
Metabolomics is considered a powerful tool to detect changes in small molecular
metabolites in organisms under different environmental stresses, which is helpful in inves-
tigating the underlying metabolic responses of the biological systems [13]. For instance,
the level of fructose-6-phosphoric, pyruvate, and creatine in Raw264.7 cells changed sig-
nificantly in the presence of H2 O2 , which further affected the amino acid and glucose
metabolism pathways [14]. Thus, it is useful to understand the information about the
metabolic profile variation, as well as the mechanism of H2 O2 -induced oxidative damage.
However, the protective effects of natural antioxidants were scarcely investigated at the
metabolic level.
Herein, the underlying mechanisms of DE against H2 O2 -induced oxidative damage
were delineated by the combination of metabolomics and lipidomics. The effects of DE
on H2 O2 -induced ROS accumulation, mitochondrial membrane potential variation, and
antioxidant-related enzyme activities were investigated. Specially, the changes of metabolic
pathways in the presence of H2 O2 or DE-H2 O2 were studied by the metabolomics and
lipidomics method for the first time. This study might provide a comprehensive under-
standing of DE in alleviating H2 O2 -induced oxidative damage.

2. Materials and Methods

2.1. Materials
Dandelion was harvested in May 2022 at Tai’an, Shandong, China. These dande-
lions were identified as the dry whole plant of Taraxacum mongolicum by Professor Xiao
Wang of Shandong University of Traditional Chinese Medicine. High glucose Dulbecco’s
Modified Eagle’s medium (DMEM) was obtained from Hyclone Laboratories (Logan, UT,
USA). 20 ,70 -dichlorofluorescin diacetate (DCFH-DA), MitoTracker Red fluorescent probe,
and Bisbenzimide H33342 trihydrochloride (Hoechst 33342) were supplied by Beyotime
Biotechnology Co., Ltd. (Shanghai, China). Undecanoic acid, 19-decanoic acid were pur-
chased from Aladdin Reagent Co., Ltd. (Shanghai, China). L-d5 -phenylalanine was bought
from Bai J&K Scientific Co., Ltd. (Beijing, China). Rat Adrenal Pheochromocytoma Cells
(PC12 cells) were bought from Procell Life Science & Technology Co., Ltd. (Wuhan, China).
Methanol and other chemical reagents were of chromatographic grade.

2.2. Preparation of DE
Dandelion was ground into powder by a disintegrator (800Y, Moling, Shanghai, China)
and sieved with 60 mesh. Then, 10 g of dandelion powder was placed in a round-bottom
flask and extracted upon refluxing twice with 60% ethanol (200 mL) for 2 h each time. The
filtrates were combined and concentrated by a rotary evaporator (EYELAN-1100, Tokyo,
Japan). The DE (1.6 g) was obtained by lyophilization.

2.3. Composition Analysis of DE

DE composition was characterized by liquid chromatography quadrupole time-of-
flight/mass spectrometer (LC-Q-TOF/MS) analysis. Liquid chromatography was per-
formed using the UHPLC system (H-Class, Waters, Milford, MA, USA). The samples were
separated on an Acquity UPLC @BEH C8 column (100 mm × 2.1, 1.7 µm, Waters, Milford,
MA, USA). The mobile phase A was water-formic acid (100:0.1, v/v) and B was acetonitrile-
formic acid (100:0.1, v/v). The gradient program was optimized as follows: 0–5 min,
5–20% A; 10–15 min, 20–24% A; 15–25 min, 24% A; 25–26 min, 24–34% A; 26–35 min,
34–40% A, 35–50 min, 40–55% A, 50–60 min, 55–70% A, 60–70 min, 70–100% A. Tandem
mass spectrometry (MS) was measured using a Q-TOF mass spectrometer equipped with an
ESI interface (Impact II, Bruker, Karlsruhe, Germany). The data were acquired in positive
and negative modes. The optimized parameters for the MS conditions were as follows: ion
spray voltage, −3000 V (−)/3500 V (+); nebulizer pressure, 2.0 bar; flow rate of dry gas,
Foods 2023, 12, 3314 3 of 14

8.0 L/min; and drying gas temperature, 200 ◦ C. The scan ranges in TOF-MS and TOF-
MS/MS modes were both m/z 50–1200. For qualitative analysis, raw LC-Q-TOF/MS data
were converted to ABF (analysis base file) format, and then processed using MS-DIAL 3.98.
Through the comparison with self-built databases of metabolomics standards, along with
analysis of the fragmentation pathways obtained from literature sources and public data,
the compounds were characterized [15].

2.4. Cell Viability Assay

PC12 cells (1 × 105 cells/well) were cultured in 96-well plates and treated with
H2 O2 (0, 100, 200, 400, 800, and 1000 µmol L−1 ), DE (0.00, 1.56, 3.12, 6.25, 12.50, 25.00,
and 50.00 µg mL−1 ), or H2 O2 (800 µmol L−1 ) in the presence of DE (12.50, 25.00, and
50.00 µg mL−1 , named as low, medium, and high concentration) for 24 h. Afterwards, 20 µL
of 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT, 0.5 mg mL−1 )
was added to each well and cultured at 37 ◦ C for another 4 h. After adding 150 µL DMSO
to each well for 10 min, the absorbance value was detected at 570 nm using a microplate
reader (Tecan, Hombrechtikon, Switzerland).

2.5. Hoechst 33342 Staining

PC12 cells (1 × 105 cells/well) were seeded in 12-well plates and treated with different
concentrations of DE for 6 h. After discarding the medium, the medium (1 mL) containing
H2 O2 (800 µmol L−1 ) was then added and stimulated for another 24 h. Finally, cells were
stained by Hoechst 33342 for 10 min and imaged by fluorescent inverted microscope (Nikon
Corp., Tokyo, Japan).

2.6. In Vitro Antioxidant Measurement

PC12 cells (1 × 105 cells/well) were cultured in 12-well plates. Then, the cells were
treated with different concentrations of DE for 6 h. After discarding the medium, the
medium (1 mL) containing H2 O2 (800 µmol L−1 ) was added and stimulated for another
24 h. ROS and MMP experiments were measured as previously reported [16]. Moreover,
superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde
(MDA) were measured by the biochemical kit (Nanjing Jiancheng Bioengineering Institute,
Nanjing, China).

2.7. Quantitative Real-Time PCR (RT-PCR) Measurement

The RT-PCR was measured by the following method of Chen et al., with minor
modification [16]. Total RNA was obtained using TRIzol reagent, and the cDNA samples
were obtained using a cDNA Synthesis Kit (Servicebio Technology Co., Ltd., Beijing, China).
The relative mRNA expression levels were quantified by a real-time PCR system using
SYBR Green qPCR Master Mix (Servicebio Technology Co., Ltd., Beijing, China), and
normalized to GAPDH expression by the 2−44Ct method. The PCR primers information is
listed in Table S1.

2.8. Metabolomics and Lipidomics Analysis

PC12 cells were cultured with H2 O2 (800 µmol L−1 ) or DE (50.00 µg mL−1 )-H2 O2
(800 µmol L−1 ) for 24 h. Later, the medium was removed and fresh medium was added and
cultured for 2 h. Then, the precooled methanol (80% v/v, −80 ◦ C) was added and incubated
for 20 min at −80 ◦ C. Undecanoic acid, 19-decanoic acid, and L-d5 -phenylalanine were
used as internal standards (5 µg/mL). After removing the cell fragment by centrifugation
for 20 min (4 ◦ C, 14,000× g), the supernatant was lyophilized. The solubilized metabolites
were filtered through a 0.22 membrane before being analyzed. As for lipidomics analy-
sis, lipids were extracted by methyl tert-butyl ether (MTBE) according to the published
method with slight modifications. Phosphatidylcholine, phosphatidylethanolamine, and
lysophosphatidylcholine were used as internal standards (5 µg/mL). Detailed conditions
for chromatography and mass spectrometry of metabolomics and lipidomics were based
on the previously reported methods [17] and are shown in the supporting information.
Foods 2023, 12, 3314 4 of 14

2.9. Statistical Analysis

Dates were expressed as mean ± standard (SD). One-way ANOVA analysis was used
to distinguish the statistical differences (p < 0.05). The multidimensional statistical analysis,
including unsupervised principal component analysis (PCA) as well as supervised partial
least squares discriminant analysis (PLS-DA), was performed through SIMCA 14.1 software.
The heatmap was completed by TB tools 6.2.

3. Results and Discussion

3.1. LC-Q-TOF/MS Characterization of DE Composition
To reveal the underlying mechanism of DE against H2 O2 -induced oxidative damage,
the DE composition was characterized by LC-Q-TOF/MS firstly (Figure S1). As displayed
in Table 1 and Figure 1A, a total of 30 chemical components, including organic acids and
flavonoids, were identified. These two types of substances are the main components that
play an antioxidant role [4]. Organic acids contained caffeic acid, 1-O-caffeoylglycerol,
p-hydroxycinnamic acid, protocatechuic acid, p-hydroxyphenylacetic acid, gallic acid,
vanillic acid, chlorogenic acid, ferulic acid, tartaric acid, chicoric acid, isochlorogenic acid
A, citric acid, caffeic acid-glucoside, shikimic acid, 3-O-feruloylquinic acid, quinic acid, and
neochlorogenic acid, while flavonoids included rutin, quercetin, luteolin, isorhamnetin,
isoquercitrin, luteolin-7-O-glucoside, apigenin, apigenin-7-O-glucoside, and naringin. Qu
et al. also found similar results, identifying 28 organic acids and 19 flavonoids [18]. The
difference in the number of compounds may be due to the different varieties of dandelion
and different detection methods. Notably, the antioxidant activities of dandelion were
mainly attributed to these organic acids and flavonoids [19].

Table 1. Identification of the components of dandelion extract.

No Component MS (m/z, Da) MS2 (m/z, Da)

1 Caffeic acid 179.0332[M-H]− 135.0357/117.0245
2 p-hydroxycinnamic acid 163.0288[M-H]− 119.0324/91.0246
3 Protocatechuic acid 153.0089[M-H]− 109.0047/91.0025
4 Isochlorogenic acid A 515.1178[M-H]− 353.0776
5 Gallic acid 169.0143[M-H]− 125.0124
6 Rutin 609.1449[M-H]− 301.0214/255.0103
7 Ferulic acid 193.0502[M-H]− 134.0203/115.0097
8 Tartaric acid 149.0092[M-H]− 87.0014
9 Neochlorogenic acid 353.0854[M-H]− 179.0247/93.0251
10 Caffeic acid-glucoside 341.0816[M-H]− 281.0649/179.0137
11 Shikimic acid 173.0421[M-H]− 110.8724
12 Chicoric acid 473.0642[M-H]− 219.0043/179.0247
13 Quinic acid 191.0558[M-H]− 93.0247
14 Vanillic acid 167.0345[M-H]− 152.0217/108.0125
15 3-O-Feruloylquinic acid 367.0124[M-H]− 193.0415/111.0386
16 p-hydroxyphenylacetic acid 151.0288[M-H]− 107.0124
17 Citric acid 191.0187[M-H]− 87.0018/67.0135
18 Chlorogenic acid 353.0834[M-H]− 191.0347/127.0298,
19 Quercetin 301.0324[M-H]− 150.9876/107.0032
20 Luteolin 285.0395[M-H]− 150.9941/133.0127,
21 Isorhamnetin 315.0506[M-H]− 300.0162/271.0034
22 Isoquercitrin 463.0864[M-H]− 301.0245/150.9934
23 Apigenin 269.0452[M-H]− 269.0353, 117.0264
24 Luteolin-7-O-glucoside 447.0928[M-H]− 285.0301
25 Apigenin-7-O-glucoside 431.0972[M+H]+ 269.0357
26 6,7-Dihydroxycoumarin 177.0124[M-H]− 149.0132/133.0178
27 Uridine 243.0614[M+H]+ 152.0237/110.0162
28 Adenosine 268.1024[M+H]+ 136.0542
29 Naringin 581.1832[M+H]+ 435.1224
30 1-O-caffeoylglycerol 253.0653[M-H]− 179.0102/133.0164
 24 Luteolin-7-O-glucoside 447.0928[M-H] 285.0301
25 Apigenin-7-O-glucoside 431.0972[M+H]+ 269.0357
26 6,7-Dihydroxycoumarin 177.0124[M-H]− 149.0132/133.0178
27 Uridine 243.0614[M+H]+ 152.0237/110.0162
28 Adenosine 268.1024[M+H]+ 136.0542
Foods 2023, 12, 3314 5 of 14
29 Naringin 581.1832[M+H]+ 435.1224
30 1-O-caffeoylglycerol 253.0653[M-H]− 179.0102/133.0164

Figure 1. (A) Chemical structures of the compounds in dandelion extract. Cell viability of PC12 cells
Figure 1. (A) Chemical structures of the compounds in dandelion extract. Cell viability of PC12 cells
after incubation with (B) H2O2, (C) DE, and (D) H2O2 (800 μmol L−1) in presence of DE for 24 h. (E)
after incubation with (B) H2 O2 , (C) DE, and (D) H2 O2 (800 µmol L−1 ) in presence of DE for 24 h.
Change in cell morphology after being treated with different groups: a, control group; b, H2O2
(E) Change
group; c, d,in
andcelle,morphology
H2O2 group inafter
thebeing treated
presence with different
of different groups: a,ofcontrol
concentrations group;
DE (12.5, b, H
25 and 502 O2
μg
group; c, d, and
mL−1); arrows e, H2 O
indicate 2 group
the in the presence
representative of bars
cells, scale different
were concentrations of DE
100 μm. Different (12.5,
letters 25 and
on the col-
50 µg mL −1 ); arrows indicate the representative cells, scale bars were 100 µm. Different letters on
umns indicate significant differences (p < 0.05). Note: in (D), − indicates cells without any treatment,
+ indicates
the columnscells treated
indicate with 800 differences
significant μmol L−1 H2(pO2<. 0.05). Note: in (D), − indicates cells without any
treatment, + indicates cells treated with 800 µmol L−1 H2 O2 .
3.2. Effects of DE on H2O2-Induced Cytotoxicity
3.2. Effects of DE on H2 O2 -Induced Cytotoxicity
The cytotoxicity evaluation was performed by MTT assay. As expected, the cell via-
bilityThe cytotoxicity
reduced evaluation
with the increasewas performed
in H by MTT assay. As expected, the cell viabil-
2O2 (Figure 1B). Noteworthily, the cell viability de-
ity reduced with the increase in H O
2 2
creased to 83% when the concentration (Figure 1B).
of H2O2 wasNoteworthily,
800 μmol L−1the cell
and viabilityadecreased
exhibited dramatic
to 83% when the concentration of H2 O2−1was 800 µmol L−1 and exhibited a dramatic drop in
drop in the presence of 1000 μmol L H2O2, indicating H2O2-induced severe oxidative
the presence of 1000 µmol L−1 H2 O2−1, indicating H2 O2 -induced severe oxidative damage to
damage to cells. Thus, 800 μmol L H2O2 were selected for the further experiments. The
cells. Thus, 800 µmol L−1 H2 O2 were selected for the further experiments. The cell viability
cell viability showed negligible change under the addition of DE, which indicates good
showed negligible change under the addition of DE, which indicates good safety and
safety and biocompatibility (Figure 1C). As shown in Figure 1D, compared to the H2O2
biocompatibility (Figure 1C). As shown in Figure 1D, compared to the H2 O2 group, the cell
viability increased in the DE-H2 O2 group, indicating that DE could alleviate H2 O2 -induced
cytotoxicity. The cell viability was 95% in DE (50.0 µg mL−1 )-H2 O2 (800 µmol L−1 ) group,
which was obviously higher than that of the H2 O2 (800 µmol L−1 ) group (83%). More-
over, the obtained results were also confirmed by the morphology changes of PC12 cells
(Figure 1E). Compared to the control group, the obvious change in cell morphology, i.e.,
abnormal sphericity, was observed after being treated with H2 O2 , and this phenomenon
was alleviated in the presence of DE. The above results suggest that DE alleviated the cell
cytotoxicity caused by H2 O2 .
 L−1) group, which was obviously higher than that of the H2O2 (800 μmol L−1) group (83%).
Moreover, the obtained results were also confirmed by the morphology changes of PC12
cells (Figure 1E). Compared to the control group, the obvious change in cell morphology,
i.e., abnormal sphericity, was observed after being treated with H2O2, and this phenome-
Foods 2023, 12, 3314 non was alleviated in the presence of DE. The above results suggest that DE alleviated
6 ofthe
14
cell cytotoxicity caused by H2O2.

3.3. Effects of DE on ROS Generation

3.3. Effects of DE on ROS Generation
Excessive generation of ROS could lead to cell apoptosis, so the concentration of ROS
Excessive generation of ROS could lead to cell apoptosis, so the concentration of ROS
was measured to assess the intervention effects of DE against H2O2-induced oxidative
was measured to assess the intervention effects of DE against H2 O2 -induced oxidative
damage [20]. DCFH-DA (an oxidation-indicative dye showing green fluorescence) was
damage [20]. DCFH-DA (an oxidation-indicative dye showing green fluorescence) was used
used to assess ROS production, and the produced green fluorescence intensity was posi-
to assess ROS production, and the produced green fluorescence intensity was positively
tively related
related to the
to the ROS ROS[21].
level level
As[21]. As shown
shown in Figure
in Figure 2, after 2, after treatment
treatment with H2with H2Ogreen
O2 , the 2, the

green fluorescence intensity in PC12 cells increased to 224%, which indicated

fluorescence intensity in PC12 cells increased to 224%, which indicated severe oxidative severe oxi-
dative damage caused by H 2O2. Compared to the H2O2 group, the green fluorescence in-
damage caused by H2 O2 . Compared to the H2 O2 group, the green fluorescence intensity
tensity decreased
decreased after
after being being pre-protected
pre-protected with different
with different concentrations
concentrations of DE,
of DE, which which de-
decreased to
creased to 169%, 149%, and 123%, respectively. This indicates that DE alleviated
169%, 149%, and 123%, respectively. This indicates that DE alleviated the ROS generation the ROS
generation
and mitigatedandthe
mitigated
occurrencethe of
occurrence
oxidativeof oxidative damage.
damage.

Figure2.2.Fluorescence
Figure Fluorescenceimages
images of PC12 cellscells
of PC12 stained with with
stained DCFH-DA
DCFH-DAafter the treatment
after of (A) DMEM
the treatment of (A)
medium (control),
DMEM medium (B) H2 O(B)
(control), 2 , (C)
H2OLow
2, (C)concentration
Low concentration of DE+H 2 O2 , 2(D)
of DE+H Medium
O2, (D) Mediumconcentration
concentration of
of DE+H
DE+H 2 O22O 2, and
, and (E)(E) High
High concentration
concentration of DE+H
of DE+H 2 O2 .2O 2. Scale
Scale barsbars
werewere 100 μm.
100 µm. (F) Relative
(F) Relative fluores-
fluorescence
cence intensity
intensity for different
for different treatmenttreatment
groups.groups.
DifferentDifferent letters indicate
letters indicate significant
significant difference
difference at p <
at p < 0.05.
0.05.
3.4. Effects of DE on MMP
3.4. Effects
Oxidativeof DEstress
on MMPcan cause mitochondrial dysfunction and reduce MMP. This is an
early Oxidative
apoptotic stress
event canandcause
can be measured bydysfunction
mitochondrial JC-1 [22]. and
JC-1reduce
aggregates
MMP.exhibit
This isred
an
fluorescence, suggesting higher MMP, while the JC-1 monomer with green
early apoptotic event and can be measured by JC-1 [22]. JC-1 aggregates exhibit red fluo- fluorescence
shows lower
rescence, MMP. The
suggesting fluorescence
higher transition
MMP, while from monomer
the JC-1 red to green
withreflects
greenthe reduction
fluorescence
in MMP [23]. As shown in Figure 3A, the
shows lower MMP. The fluorescence transition2 from H O 2 group induced an obvious reduction
red to green reflects the reduction in
in MMP, as confirmed by the strongest green fluorescence and weakest
MMP [23]. As shown in Figure 3A, the H2O2 group induced an obvious reduction in MMP, red fluorescence.
This finding was
as confirmed quantitatively
by the displayed
strongest green in the fluorescence
fluorescence and weakestintensity plots (Figure
red fluorescence. This3B,C).
find-
After addition of DE, the red/green fluorescence intensity ratio was obviously
ing was quantitatively displayed in the fluorescence intensity plots (Figure 3B,C). After increased
compared
addition oftoDE, the the
H2 Ored/green
2 group, especially at the
fluorescence high concentration
intensity of DE (Figure
ratio was obviously 3D). com-
increased This
indicates that DE alleviated the reduction of MMP caused by H 2 O 2 and
pared to the H2O2 group, especially at the high concentration of DE (Figure 3D). This in-mitochondrial
damage. The results were consistent with the tendency of the ROS analysis.
dicates that DE alleviated the reduction of MMP caused by H2O2 and mitochondrial dam-
age.Effects
3.5. The results
of DE onwere consistent with the
Antioxidant-Related tendency
Enzyme of the ROS analysis.
Activities
Superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) are important
antioxidant enzymes in human body, which can effectively scavenge excess oxygen-free
radicals, and maintain cell vitality [24]. As shown in Figure 4A,B, the levels of SOD
and GSH-Px reduced in H2 O2 group. After the cells were pre-incubated with different
concentrations of DE, the levels of SOD and GSH-Px increased in a dose-dependent man-
ner. Meanwhile, the mRNA expression of SOD1 and GSH-Px showed the same trend
(Figure 4C,D). Moreover, malondialdehyde (MDA), reflecting the degree of cell damage,
was also detected by the assay kit. Compared to control group, the level of MDA increased
significantly in H2 O2 group, while decreased upon the addition of DE (Figure S2). Collec-
 Foods 2023, 12, 3314 7 of 14

Foods 2023, 12, x FOR PEER REVIEW 7 of 14

tively, DE exhibited superior antioxidant abilities and were effective at alleviating H2 O2
oxidative stress.

Figure
Figure3.3.Fluorescence
Fluorescenceimaging
imagingofofthe
themitochondrial
mitochondrialmembrane
membranepotential
potentialfor
for(A)
(A)PC12
PC12cells
cellsstained
stained
with
withJC-1
JC-1before
before(control)
(control)and
andafter
aftertreatment
treatmentwith
with(H(H2O
2O2), ),
2 low,
low,medium,
medium,andandhigh
highconcentration
concentrationofof
DE.
DE.Scale
Scalebars
barswere
were100
100μm.
µm.The
Thefluorescence
fluorescenceintensity
intensityofof(B)(B)red
redfluorescence
fluorescenceand
and(C)
(C)green
greenfluo-
fluo-
Foods 2023, 12, x FOR PEER REVIEWrescence. (D) Red/green fluorescence intensity ratio. Different letters indicate significant difference 8 of 14
rescence. (D) Red/green fluorescence intensity ratio. Different letters indicate significant difference
at p < 0.05.
at p < 0.05.

3.5. Effects of DE on Antioxidant-Related Enzyme Activities

Superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) are important an-
tioxidant enzymes in human body, which can effectively scavenge excess oxygen-free rad-
icals, and maintain cell vitality [24]. As shown in Figure 4A,B, the levels of SOD and GSH-
Px reduced in H2O2 group. After the cells were pre-incubated with different concentra-
tions of DE, the levels of SOD and GSH-Px increased in a dose-dependent manner. Mean-
while, the mRNA expression of SOD1 and GSH-Px showed the same trend (Figure 4C,D).
Moreover, malondialdehyde (MDA), reflecting the degree of cell damage, was also de-
tected by the assay kit. Compared to control group, the level of MDA increased signifi-
cantly in H2O2 group, while decreased upon the addition of DE (Figure S2). Collectively,
DE exhibited superior antioxidant abilities and were effective at alleviating H2O2 oxidative
stress.

Figure4.
Figure Thelevel
4.The level of
of (A)
(A) SOD
SOD and
and (B)
(B) GSH-Px
GSH-Px inindifferent
differenttreated
treatedgroups.
groups.The
ThemRNA
mRNAexpressions
expressionsof
(C) SOD1 and (D) GSH-Px in different treated groups. Different letters indicate significant difference
of (C) SOD1 and (D) GSH-Px in different treated groups. Different letters indicate significant differ-
at p <at0.05.
ence p < 0.05.

3.6. Effects of DE on Metabolic Response

3.6. Effects of DE on Metabolic Response
The targeted metabolomics and lipidomics approaches were performed to evaluate
The targeted metabolomics and lipidomics approaches were performed to evaluate
the effects of DE on H2 O2 -induced oxidative damage at the metabolic level. The metabolic
the effects of DE on H2O2-induced oxidative damage at the metabolic level. The metabolic
response of PC12 cells was evaluated after being treated with H2 O2 (800 µmol L−−11 ) and
response of PC12 − cells was evaluated after being treated with H2O2 (800 μmol L ) and
H2 O2 (800 µmol L−1 1 ) in the presence of DE (50 µg mL− 1 ) (DE-H O2 ). Principal component
H2O2 (800 μmol L ) in the presence of DE (50 μg mL ) (DE-H22O2).
−1 Principal component
analysis (PCA) score plots indicated that all QC samples were tightly clustered, suggesting
the robustness of the capture method and the reliability of the data collected during the
sample analysis (Figure S3A,B). Next, PLS-DA was established to illustrate the different
responses among the control, H2O2, and DE-H2O2 groups. As illustrated in Figure S3C,D,
 Foods 2023, 12, 3314 8 of 14

analysis (PCA) score plots indicated that all QC samples were tightly clustered, suggesting
the robustness of the capture method and the reliability of the data collected during the
sample analysis (Figure S3A,B). Next, PLS-DA was established to illustrate the different
responses among the control, H2 O2 , and DE-H2 O2 groups. As illustrated in Figure S3C,D,
the DE-H2 O2 group presented an apparent separation from the control group and H2 O2
group for metabolome and lipidomics, suggesting that H2 O2 treatment significantly dis-
rupted cellular metabolic processes, and that the addition of DE changed H2 O2 -metabolic
disorders. Moreover, the model parameters (prediction ability (Q2 ) and credibility (R2 ))
for the metabolome were 0.757 and 0.901, and the Q2 and R2 for lipidomics were 0.809
and 0.987, respectively, suggesting satisfactory prediction and faithful representation. The
permutation test was established to validate PLS-DA models (n = 200). It is shown in
Figure S2E that vector values of R2 and Q2 obtained in the permutation test were lower
than the original value, indicating that the established model for the metabolome was
stable and reliable. Similar results were also observed for lipidomics (Figure S3F), which
showed that the obtained vector values R2 and Q2 were smaller than the original values,
indicating that no overfitting occurred in the PLS-DA score plot of lipidomics.
Figure 5 illustrates the change of the different metabolites and lipids after being treated
with H2 O2 or DE-H2 O2 . It was revealed that 36 metabolites were obviously changed in
the H2 O2 or DE-H2 O2 group based on screening criteria (p < 0.05) (Figure S4A), which
were involved in glycolysis, the citric acid cycle (TCA cycle), antioxidant capacity, the
central nervous system, energy metabolism, and amino acid metabolic pathways. Further-
more, enrichment analysis (based on p < 0.05) was used to study the effects of H2 O2 or
DE- H2 O2 on various cellular metabolic pathways. Compared to the control group, H2 O2
R PEER REVIEW treatment significantly affected glycolysis, nicotinate and nicotinamide metabolism,9 of 14
serine
and threonine metabolism, and phenylalanine, glycine, and pyruvate metabolism. After
being pre-treated with DE, the disordered pathways induced by H2 O2 were improved, and
only phenylalanine, tyrosine and tryptophan biosynthesis were significantly disrupted
sphingomyelin (SM), fatty
(Figure acids
S4B). As for(FA), ceramidesphosphatidylcholine
lipid metabolites, (Cer), triglycerides
(PC), (TG), diglycerides
phosphatidylethanolamine
(DG), hexosylceramides (HexCer), (SM),
(PE), sphingomyelin alkylfatty
andacids
alkenyl substituent
(FA), ceramides (Cer),PCs (PC-O),(TG),
triglycerides alkyl and
diglycerides
alkenyl substituent PEs (PE-O) and cholesterol esters (CE) were significantly changed af- and
(DG), hexosylceramides (HexCer), alkyl and alkenyl substituent PCs (PC-O), alkyl
alkenyl substituent PEs (PE-O) and cholesterol esters (CE) were significantly changed after
ter different treatments (Figure 5B).
different treatments (Figure 5B).

Figure 5. Heatmap of relative content of different metabolites for (A) metabolomics and lipids for
Figure 5. Heatmap of relative content of different metabolites for (A) metabolomics and lipids for
(B) lipidomics analysis after being treated with H2 O2 or DE-H2 O2 .
(B) lipidomics analysis after being treated with H2O2 or DE-H2O2.

3.7. Effects of DE on Energy Metabolism

The results from metabolism analysis indicated that energy metabolism, including
glycolysis, TCA cycle, nicotinate and nicotinamide metabolism, was obviously disturbed
 Foods 2023, 12, 3314 9 of 14

3.7. Effects of DE on Energy Metabolism

The results from metabolism analysis indicated that energy metabolism, including
glycolysis, TCA cycle, nicotinate and nicotinamide metabolism, was obviously disturbed
after intervention by H2 O2 (Figure 6). For example, the intermediate metabolites (includ-
ing glucose-6-phosphate, fructose-1,6-diphosphate, and pyruvic acid) were significantly
changed. As one of the key limit-rate reactions in glycolysis, hexokinase catalysis can
convert glucose into glucose-6-phosphate [25]. The relative content of glucose-6-phosphate
in the H2 O2 group was 4.8-fold higher than that in the control group, while it reduced by
14% in the presence of DE. Meanwhile, fructose 6-phosphate is catalyzed by phosphofruc-
tokinase to produce fructose 1,6-diphosphate, which is the second rate-limiting reaction in
glycolysis [26]. The variation of the relative content of fructose-1,6-diphosphate exhibited a
similar trend with glucose-6-phosphate after DE treatment. Moreover, as a direct carbon
precursor for the synthesis of acetyl-CoA, pyruvate also exerts an essential role in the
TCA cycle. The level of pyruvate significantly increased after treatment with H2 O2 , being
5.75-fold higher than that in control group. After being pre-protected with DE, the relative
content of pyruvate decreased by 4.3% compared to H2 O2 group. All these results suggest
s 2023, 12, x FOR PEER REVIEW 10 of 14
that glycolysis was perturbed when oxidative damage occurred and that DE could alleviate
the disturbance of glucose metabolism induced by H2 O2 .

Changes pathway
Figureof6.metabolic
Figure 6. Changes of metabolic
afterpathway after with
being treated beingHtreated
2O2 or Hwith H2the
2O2 in O2 or H2 O2 in
presence of the presence
DE. “*” meansof significant differences
DE. “*” means (p < 0.05).
significant “#” means
differences significant
(p < 0.05). differences
“#” means (p < 0.01).
significant “ns” (p < 0.01).
differences
means two columns havetwo
“ns” means no difference.
columns have no difference.

3.8. Effects of DE onThe TCA Acid

Amino cycleMetabolism
participates in physiological processes, providing energy and building
blocks for cells to sustain
Amino acids are fundamental constituentscell survival
of [27]. Compared
proteins, to theancontrol
which play group, the level of
irreplaceable
oxaloacetate increased, indicating the disruption of TCA cycle
role in energy metabolism [30]. The amino acid metabolism was disrupted after treatment after H 2 O2 addition. The
level of oxaloacetate decreased after being treated with DE, which
with H2O2 (Figure 6). The relative content of phenylalanine decreased by 26.58% com- indicated the protective
pared with the control group. Reversely, the level of phenylalanine increased by 20.2%[28]. Notably,
effect of DE against oxidative damage via the down-regulation of oxaloacetate
nicotinate and nicotinamide metabolism were also disordered in the H2 O2 group and
upon the addition of DE. As an indirect indicator of glycolysis, phenylalanine could influ-
reversed after being treated with DE. Nicotinate was metabolized to nicotinamide, a
ence the extracellular acidification rate (ECAR), which could reflect the activities of gly-
precursor of nicotinamide adenine dinucleotide (NAD+), which prevents oxidative damage
colysis [31]. These results demonstrate that DE could alleviate H2O2-induced glycolysis
by regulating energy metabolism [29]. All these results suggest that glycolysis, TCA cycle
disorder. Moreover, the level of acetyl-lysine was up-regulated after being treated with
nicotinate and nicotinamide metabolism were perturbed when oxidative damage occurred
H2O2, indicating the cells suffered oxidative damage. Acetyl-lysine was down-regulated
and that DE could alleviate H2 O2 -induced metabolic disorder.
to the normal level in the presence of DE, which was in line with the previous results [32].

3.9. Effects of DE on Other Metabolism Pathways

Studies have reported that tryptophan is involved in the regulation of oxidative
stress, inflammation, and the immune response [33]. In addition, kynurenic acid is the
Foods 2023, 12, 3314 10 of 14

3.8. Effects of DE on Amino Acid Metabolism

Amino acids are fundamental constituents of proteins, which play an irreplaceable
role in energy metabolism [30]. The amino acid metabolism was disrupted after treatment
with H2 O2 (Figure 6). The relative content of phenylalanine decreased by 26.58% compared
with the control group. Reversely, the level of phenylalanine increased by 20.2% upon the
addition of DE. As an indirect indicator of glycolysis, phenylalanine could influence the
extracellular acidification rate (ECAR), which could reflect the activities of glycolysis [31].
These results demonstrate that DE could alleviate H2 O2 -induced glycolysis disorder. More-
over, the level of acetyl-lysine was up-regulated after being treated with H2 O2 , indicating
the cells suffered oxidative damage. Acetyl-lysine was down-regulated to the normal level
in the presence of DE, which was in line with the previous results [32].

3.9. Effects of DE on Other Metabolism Pathways

Studies have reported that tryptophan is involved in the regulation of oxidative
stress, inflammation, and the immune response [33]. In addition, kynurenic acid is the
downstream metabolite of tryptophan, which also plays an indispensable role in protecting
the central nervous system [34]. As displayed in Figure 6, the levels of tryptophan and
kynurenic acid were obviously changed after being treated with H2 O2 , indicating that
the central nervous system was damaged. Reversely, the addition of DE mitigated the
nervous system damage via participating in the metabolism pathway related to antioxidant.
Bioactive compounds including creatine and its phosphate form (creatinine) were also
changed after being treated with H2 O2 and recovered to be almost equal to the control level
in the presence of DE [35]. The above results demonstrate that oxidative stress occurred
after being treated with H2 O2 and was alleviated by the addition of DE.

3.10. Effects of DE on Lipid Metabolism

Extensive studies have identified bioactive compounds derived from fruit extracts and
herbal products that could modulate lipid metabolism in a positive manner [36,37]. Thus,
the effects of DE on H2 O2 -induced lipid metabolism disorders were elaborated (Figure 7).
The transformation of SM to Cer is involved in cell apoptosis [38]. Compared to the
control group, the level of SM was decreased by 32.3% and the level of Cer was increased
by 70% after treatment with H2 O2 , indicating the apoptotic signal was active at these
conditions. Phosphatides, including PC and PE, are important structural foundations of
cell membranes. They participate in the cytidine diphosphate (CDP)-diacylglycerol and 1,2-
diacylglycerol (DAG) pathways and are adjusted by various enzymes [39]. The PC content
was increased after treatment with H2 O2 , illustrating that the cell membrane integrity was
destroyed. Meanwhile, the intracellular glycerophosphatidylcholine also showed a change
trend similar to that of PC. A previous study reported that glycerophosphatidylcholine is a
metabolite of lipid oxidation and is also associated with the integrity of cell membranes [40].
Compared to the control group, the level of PE was decreased by 38.1% in the H2 O2 group
and was restored to the normal level in the presence of DE. LPC and LPE are the degradation
products obtained by removing one molecule of fatty acid from PC and PE, respectively.
In the oxidative stress state, the level of LPC and LPE increased, demonstrating that the
catabolism of PC and PE was enhanced. The addition of DE alleviated the relative levels of
PC and PE back to normal.
As an ether-containing phospholipid, plasmalogens play an important role in main-
taining cell vitality and normal cell metabolism [41]. The disorder of plasmalogens is
regarded as an indicator of oxidative stress. In the current study, the PC-O and PE-O were
changed after incubation with H2 O2 , and the addition of DE was found to be able to allevi-
ate this disorder (Figure S5). The change of plasmalogens was also found in As3+ -induced
oxidative stress, which further proved this finding [42].
 choline is a metabolite of lipid oxidation and is also associated with the integrity of cell
membranes [40]. Compared to the control group, the level of PE was decreased by 38.1%
in the H2O2 group and was restored to the normal level in the presence of DE. LPC and
LPE are the degradation products obtained by removing one molecule of fatty acid from
PC and PE, respectively. In the oxidative stress state, the level of LPC and LPE increased,
Foods 2023, 12, 3314 demonstrating that the catabolism of PC and PE was enhanced. The addition of DE alle- 11 of 14
viated the relative levels of PC and PE back to normal.

Figure 7. The change of lipid metabolic pathway after being treated with H2O2 or H2O2 in presence
Figure 7. The change of lipid metabolic pathway after being treated with H2 O2 or H2 O2 in presence
of DE. “*” means significant differences (p < 0.05). “#” means significant differences (p < 0.01). “ns”
of DE. “*” means significant differences (p < 0.05). “#” means significant differences (p < 0.01).
means two columns have no difference.
“ns” means two columns have no difference.
As an ether-containing phospholipid, plasmalogens play an important role in main-
Triglyceride (TG) is an important storage lipid in cells to load excessive fatty acid. TG
taining cell vitality and normal cell metabolism [41]. The disorder of plasmalogens is re-
accumulation caused by oxidative damage could be found in stressed cells [43]. Currently,
garded as antheindicator of oxidative
level of TG in the H2 Ostress. In the current study, the PC-O and PE-O were
2 group was 1.02-fold higher than in the control group. DG and FA
changed after incubation with H 2O2, and the addition of DE was found to be able to alle-
are direct precursors of TG synthesis, which exhibited similar trends to TG under the same
viate this disorder (Figure
conditions [44]. S5).
AfterThe change
being of with
treated plasmalogens wasofalso
DE, the level TG,found
DG, asinwell
As3+as
-in-
FA decreased,
duced oxidative stress, which further proved this finding [42].
implying that the addition of DE alleviated the H2 O2 -induced lipid metabolism disorder.
Triglyceride (TG) is an important storage lipid in cells to load excessive fatty acid. TG
accumulation 4. caused by oxidative damage could be found in stressed cells [43]. Currently,
Conclusions
the level of TG inInthe H2O2 group
summary, was 1.02-fold
the underlying higher than
mechanism in the
of DE control
against H group. DG and
O -induced oxidative dam-
2 2
age was investigated at the metabolic level for the first time. Cell viability indicated that
DE alleviated H2 O2 -induced cytotoxicity. In addition, DE efficiently protected PC12 cells
against H2 O2 -induced oxidative damage by inhibiting ROS accumulation, increasing the
MMP, and improving the antioxidant-related enzyme activities. Importantly, the pres-
ence of DE decreased the H2 O2 -induced energy metabolism, lipid metabolism, central
nervous system and antioxidant-related metabolism disorder, thus alleviating oxidative
damage. The results are helpful in clarifying the underlying mechanism of DE against
H2 O2 -induced oxidative damage and provide new insights into dandelion as a suitable
additive for functional food products.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/foods12173314/s1, Figure S1: Total ion chromatogram of mass
spectrometer of DE. Figure S2: The level of MDA in different treated groups. Different letters
indicate significant difference at p < 0.05; Figure S3: The metabolic profiles of PC12 cells under
different treatments. PCA score plots for (A) metabolomics and (B) lipidomics, PLS-DA score plots for
(C) metabolomics for (D) lipidomics, a total of 200 random permutation tests of the PLS-DA model
for (E) metabolomics and (F) lipidomics; Figure S4: Enrichment analysis of differential metabolites in
PC12 cells under different treatments. (A) Effects of H2 O2 on metabolic pathways in comparation
Foods 2023, 12, 3314 12 of 14

with the control, (B) effects of DE-H2 O2 on metabolic pathways in comparation with the control;
Figure S5: The relative level of PE-O (A) and PC-O (B) in PC12 cells after being treated with H2 O2 or
H2 O2 in the presence of DE; Table S1: Primer sequences used for RT-PCR.
Author Contributions: Conceptualization, Y.C. and M.T.; methodology, S.F. and X.Y.; validation,
X.Y.; investigation, Y.C.; writing—original draft preparation, Y.C.; writing—review and editing,
M.T.; funding acquisition, M.T. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by the National Science Fund for Distinguished Young Scholars
of China (31925031).
Institutional Review Board Statement: Rat Adrenal Pheochromocytoma Cells (PC12 cells) were
bought from Procell Life Sci-ence & Technology Co., Ltd. (Wuhan, China). Studies not involving
humans or animals.
Informed Consent Statement: Studies not involving humans or animals.
Data Availability Statement: The datasets generated for this study are available on request to the
corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.

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