Journal of Pharmaceutical and Biomedical Analysis 242 (2024) 116019

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Journal of Pharmaceutical and Biomedical Analysis

journal homepage: www.journals.elsevier.com/journal-of-pharmaceutical-and-biomedical-analysis

UPLC-QTOF-MS based metabolomics unravels the modulatory effect of

ginseng water extracts on rats with Qi-deficiency
Yanyi Li a, Yi Wu a, *, Hanlin Li a, Meiyuan Wang a, Yang Gao a, Shuhua Pei a, Shu Liu b,
Zhiqiang Liu b, Zhongying Liu a, Lihui Men c, *
a
School of Pharmaceutical Sciences, Jilin University, Changchun 130021, PR China
b
National Center of Mass Spectrometry in Changchun & Jilin Provincial Key Laboratory of Chinese Medicine Chemistry and Mass Spectrometry, Changchun Institute of
Applied Chemistry, Chinese Academy of Sciences, Changchun 130021, PR China
c
College of Basic Medical Sciences, Jilin University, Changchun 130021, PR China

A R T I C L E I N F O A B S T R A C T

Keywords: Ginseng is commonly used as a nutritional supplement and daily wellness product due to its ability to invigorate
Ginseng water extract qi. As a result, individuals with Qi-deficiency often use ginseng as a health supplement. Ginsenosides and
Ginsenoside polysaccharides are the primary components of ginseng. However, the therapeutic effects and mechanisms of
Ginseng polysaccharide
action of these components in Qi-deficiency remain unclear. This study aimed to determine the modulatory
Qi-deficiency
Metabolomics
effects and mechanisms of ginseng water extract, ginsenosides, and ginseng polysaccharides in a rat model of Qi-
TLR4/NF-κB deficiency using metabolomics and network analysis. The rat model of Qi-deficiency was established via
swimming fatigue and a restricted diet. Oral administration of different ginseng water extracts for 30 days
primarily alleviated oxidative stress and disrupted energy metabolism and immune response dysfunction caused
by Qi-deficiency in rats. Ultra-high-performance liquid chromatography combined with quadrupole time-of-
flight mass spectrometry (UPLC-QTOF-MS) was used for untargeted serum metabolomic analysis. Based on the
analysis results, the active constituents of ginseng significantly reversed the changes in serum biomarkers related
to Qi-deficiency in rats, particularly energy, amino acid, and unsaturated fatty acid metabolism. Furthermore,
analysis of the metabolite-gene network suggested that the anti-Qi-deficiency effects of the ginseng components
were mainly associated with toll-like receptor (TLR) signaling and inflammatory response. Additional verifica­
tion revealed that treatment with the ginseng components effectively reduced the inflammatory response and
activation of the myocardial TLR4/NF-κB pathway induced by Qi-deficiency, especially the ginseng water ex­
tracts. Therefore, ginseng could be an effective preventive measure against the progression of Qi-deficiency by
regulating metabolic and inflammatory responses.

1. Introduction multipathway characteristics [6,7]. Owing to its efficacy and excellent

According to Chinese medicine theory, Qi-deficiency is a lack of vital
energy [1]. Qi-deficiency can be caused by congenital immune defi­
ciency, malnutrition, excessive fatigue, failure to recover after surgery,
and frailty due to old age [2]. The main clinical manifestations of
Qi-deficiency include pale complexion, limb weakness, dizziness, and
night sweats [3]. In modern medicine, this condition is commonly
referred to as subhealth [4]. Owing to the chronic and complex nature of
Qi-deficiency, prolonged utilization of novel multitarget therapies is
necessary [5]. Traditional herbal medicines have a long history of
clinical use and are known for their multicomponent, multitarget, and
safety, traditional Chinese medicine is increasingly employed in the
clinics to treat disorders.
Ginseng, a tonic popular worldwide, has been used for centuries.
Ginseng is known for its ability to nourish the spleen and lungs,
replenish fluids and blood, calm nerves, and enhance intelligence.
However, ginseng is primarily used to treat qi and blood deficiency in
five internal organs, particularly in conditions related to the spleen,
lung, kidney, heart, and other Qi-deficiency syndromes. With contin­
uous advancements in modern medicine and a growing awareness of
human health, the methods of ginseng consumption have been diversi­
fied. In addition to traditional decoctions and infusions, ginseng can now

* Corresponding authors.
E-mail addresses: wuyi@jlu.edu.cn (Y. Wu), menlh@jlu.edu.cn (L. Men).

https://doi.org/10.1016/j.jpba.2024.116019
Received 30 November 2023; Received in revised form 29 January 2024; Accepted 5 February 2024
Available online 7 February 2024
0731-7085/© 2024 Elsevier B.V. All rights reserved.
Y. Li et al.
be consumed after soaking in water or wine or grinding into a powder.
Currently, the most prevalent methods of ginseng consumption include
dietary supplements and functional foods for daily health maintenance
[8]. Ginseng is renowned for its various functions, including enhancing
vitality, strengthening pulse, invigorating the spleen, and benefiting the
lungs, thereby promoting body fluids, nourishing blood, soothing
nerves, and enhancing wisdom [9]. Ginseng has a significant effect on
Qi-deficiency syndrome in the spleen, lungs, kidneys, and heart [10].
Water extracts of ginseng, ginsenosides, and ginseng polysaccharides
have medicinal value [11–13]. Lin et al. [3] revealed that ginseng has a
therapeutic effect on Qi-deficiency. However, the specific benefits of the
bioactive components of ginseng in rats with Qi-deficiency and the key
components responsible for their protective effects have not been suf­
ficiently illustrated.
Metabolomics is a field in systems biology that involves detecting
and analyzing variations in endogenous molecules in response to in­
ternal and external factors using different samples [14]. Recently,
metabolomics has been extensively used to characterize and illustrate
the beneficial roles of functional foods in health promotion and disease
prevention [15,16]. With the development of analytical technology and
bioinformatic approaches, functional food-related metabolomics
research has become increasingly available and effective [17,18]. Based
on an ultra-high performance liquid chromatography coupled with
quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS)
approach, Zhang et al. [19] revealed the metabolic management effect
of Poria cocos aqueous extract on rats with chronic sleep deprivation,
which subsequently modulated anxiety-like behavior. Overall, metab­
olomics may provide insights into the health benefits of different
ginseng water extracts, which are mainly used to treat Qi-deficiency.
The aim of this study was to determine the effects of different ginseng
active components on Qi-deficiency in vivo and to understand the cor­
responding mechanisms. The functional and metabolic effects of ginseng
extract on the serum metabolic profiles of rats with Qi-deficiency were
analyzed using a UPLC-QTOF-MS-based non-targeted metabolomics
approach. In addition, bioinformatics analysis combined with western
blot analysis was conducted to further illustrate and validate the
mechanisms of ginseng extract in Qi-deficiency syndrome. Overall, these
findings enhance our understanding of the functional mechanisms of
ginseng components in Qi-deficiency syndrome.
2. Materials and methods
2.1. Preparation of ginseng water extracts
The acquisition of ginseng was accomplished through purchase from
Fusong (Jilin, China) and authentication by Professor Zhiqiang Liu
(Changchun Institute of Applied Chemistry, Chinese Academy of Sci­
ences, Changchun, China). The voucher specimens were deposited in
Changchun Institute of Applied Chemistry, Chinese Academy of Sci­
ences, Changchun, China (No. YHZY20140508).
Cut ginseng into 2–3 mm slices, weigh 1 kg of ginseng, soak in 8
times the volume (m/v) of water for 1 h, heat to reflux for 1 h, and filter
the extract with 3 layers of gauze. Repeat 3 times, combine the extracts,
and dry to obtain the water extract of ginseng. After the extract was
obtained according to the above process, it was concentrated under
reduced pressure to 2000 mL, precipitated with 80% ethanol for 24 h,
filtered with suction, and the solid was directly put into a vacuum drying
oven to dry to obtain the total polysaccharide ginseng. The supernatant
was concentrated under reduced pressure to a specific gravity of 1:1, and
then loaded onto D101 macroporous resin to prepare total ginsenosides.
D101-Macroporous resin saponin purification process: first soak the
macroporous resin with 95% ethanol overnight, wash with water until
there is no alcohol smell, and pack it into a column. After washing with 6
times the column volume of water, the concentrated solution was slowly
added to the top of the resin and adsorbed overnight. First rinse with 4
times the volume of pure water, then rinse with 8 times the volume of
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Journal of Pharmaceutical and Biomedical Analysis 242 (2024) 116019
70% ethanol, and collect the alcohol washing solution. After concen­
trating under reduced pressure, put it into a vacuum oven for drying to
obtain total ginsenosides.
The total polysaccharide content was determined by anthrone sulfate
reaction colorimetry (Supplementary Method S1). The results showed
that the polysaccharide content in the total polysaccharide fraction of
ginseng was 51.84% (w/w), and the polysaccharide content in the water
extract of ginseng accounted for 64.5% (w/w). The content of total sa­
ponins was determined by vanillin perchloric acid reaction colorimetry
(Supplementary Method S2). The results showed that the content of
saponin in the water extract of ginseng was 9.28% (w/w), and the
content of saponin in the total saponin fraction of ginseng was 50.47%
(w/w).
2.2. Animals and model construction
A total of 50 male Wistar rats, weighting 150–180 g, were obtained
from Liaoning Changsheng biotechnology co., Ltd. (Liaoning, China;
Certificate no. SCXK 2020–0001). All animals were kept in a barrier
system with regulated humidity (50 ± 5%) and temperature (21 ± 2 ℃),
and under light-dark cycle of 12 h every day; then, acclimated for 7 days.
All the animal studies were performed according to institutional
guidelines for the care and use of laboratory animals. Protocols were
approved by the Laboratory Animal Ethics & Welfare Committee in
College of Basic Medical Sciences of Jilin University (Approved No.
2021015).
Rats were randomly divided into 5 groups, namely normal control
group (Con), Qi-deficiency model group (Model), ginseng water extract
(GE) treatment group, ginseng saponin (GS) treatment group, and
ginseng polysaccharide (GP) treatment group, with 10 rats in each
group. The model was established with reference to the experimental
methods of Lin et al. [3]. The Qi-deficiency rat model was established by
inducing swimming fatigue and implementing a restricted diet. Rats in
both the model group and the ginseng administration groups were
subjected to swimming in a tank that was 50 cm deep, at a temperature
of 20–25 ◦ C. The swimming continued until the rats reached exhaustion,
with the load being equivalent to 10% of their body weight (10 s after
rats could not reach the surface of water) for 15 days. The swimming
time was recorded every day. These rats were also fed with a restrained
diet (standard food weight of 5% body weight). While rats in the Con
group were fed free with standard food. Rats were weighted every 7
days. We conducted model evaluation on rats. The results revealed that
the rats in the model group exhibited several noticeable differences
compared to the normal rats. These differences included decreased hair
luster, fatigue, drowsiness, soft feces, blood routine disorders, abnormal
energy metabolism, damaged immune system, and oxidative damage.
Importantly, these symptoms were consistent with those observed in rats
with Qi-deficiency (Supplementary method S3). After swimming, rats
were continuous oral administrated with GE, GS and GP for 30 days. The
therapeutic effects of different dosages were verified through pre­
liminary experiments, and the final dosage was determined to be 0.45
g/kg. (Supplementary method S4), and the dosage was calculated based
on ginseng water extract. The Con group and model group were given
the same volume of physiological saline every day. At the end of the
experiment, all rats were fasted for 12 h and then sacrificed.
2.3. Sample collection and preparation
After the rats were sacrificed, blood, thymus and spleen samples
were collected. Each rat was given 1 mL of blood using an anticoagulant
collection vessel and the remaining blood was immediately centrifuged
at 4 ◦ C at 3000 rpm for 10 min. The centrifuged serum was collected in
aliquots and stored at − 80 ◦ C for subsequent biochemical analysis. The
spleen and thymus were weighed and calculate the organ coefficients.
The rat heart and thigh muscles were obtained and stored at − 80 ◦ C for
subsequent biochemical index detection.
Y. Li et al.
2.4. Biochemical parameters analysis
The anticoagulant blood was packed in an ice box and sent to the
animal hospital for routine blood testing. The rat heart tissue, thigh
muscle tissue and PBS solution were homogenized at a ratio of 1:9 (g/
mL). After fully homogenized, the homogenate was centrifuged at 3000
rpm for 20 min at 4 ◦ C, and the supernatant was taken. Serum samples
and tissue homogenate supernatant samples were stored at 4 ◦ C prior to
testing. ELISA kits (Biotechnology Co, LTD, Shanghai, China) were
applied to evaluate the content of TNF-α, IL-1β, IL-6, INF-γ, MDA, and
the activities of LDH, ATPase, CK, SOD in serum of tissues.
2.5. Preparation of serum samples for UPLC-QTOF-MS analysis
Before UPLC-QTOF-MS analysis, 100 µL of serum samples were taken
and thawed at 4 ◦ C, methanol and serum samples were mixed at a ratio
of 4:1, vortexed for 1 min, placed in a refrigerator at 4 ◦ C for 20 min, and
then centrifuged at 15,000 rpm at 4 ◦ C for 15 min. The supernatant was
taken and filtered through a 0.22 µm filter for LC-MS analysis. Since the
elution order and retention times in the analytical system may change
during LC-MS analysis, it is necessary to monitor the consistency of the
system. In this study, 30 µL serum samples were pooled to obtain quality
control (QC) samples for method validation. A QC sample was run 12
times at the beginning of the entire sample list and injected every 10
samples during the analytical run to verify system consistency.
2.6. UPLC-QTOF-MS conditions
Serum samples were analyzed using a Waters Acquity UPLC system
in conjunction with a Q-TOF SYNAPT G2 mass spectrometer (Waters,
USA). Separation of the samples was achieved through the use of a
Waters ACQUITY UPLC BEH C18 Column (1.7 µm, 2.1 mm × 50 mm;
Waters) maintained at a temperature of 35 ◦ C and an injection volume of
5 µL. The mobile phase consisted of 0.1% formic acid (v/v) in water for
mobile phase A and acetonitrile for mobile phase B, with both mobile
phases containing 0.1% formic acid (v/v) in water and acetonitrile,
respectively. The flow rate was 0.3 mL/min. The elution gradient of B
was from 5% B to 100% B in 11 min, 100–5% B in 11 min - 11.1 min, and
then held at 5% B for 4 min. All samples were kept at 4 ◦ C during
analysis. In positive ion mode, the capillary voltage, cone voltage, and
extraction cone voltage were 3.0 kV, 35 V, and 5.0 V, respectively. In
negative ion mode, the capillary, cone, and extraction cone voltages
were 2.0 kV, 35 V, and 5.0 V, respectively. ESI sources in positive and
negative ion modes were used in MS analysis. MS data was collected in
full scan mode with a mass range of 50–1000 Da and a scan time of 0.5 s.
Sodium formate is applied to set up mass spectrometer calibration.
Leucine enkephalin at 2 ng/mL was used as the reference mass, and the
spray injection flow was locked at 5 µL/min to ensure accuracy during
MS analysis. MSE is used for MS2 analysis with a low collision energy of
6 eV and a high collision energy of 25–35 eV.
2.7. Data acquisition and analysis
UPLC-MS raw data were processed using MassLynx V4.1 software
(Waters Corporation, Milford, USA). Raw data were processed for
alignment peak picking, deconvolution and identification and normali­
zation using Progenesis QI software. The data processed by QI software
were imported into EZinfo 2.0 software for principal component analysis
(PCA) and orthogonal projection to latent structure square discriminant
analysis (OPLS-DA). Compounds with VIP values (VIP > 1) and t-test (P
< 0.05) were considered as the potential biomarkers. Potential markers
were identified using biochemical databases such as HMDB
(http://www.hmdb.ca/) and KEGG (http://www.kegg.com/). Pathway
enrichment analysis was performed by MetaboAnalyst (https://www.
metaboanalyst.ca/) to investigate the biological characteristics of the
biomarkers. The metabolite-gene networks were analyzed through
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Journal of Pharmaceutical and Biomedical Analysis 242 (2024) 116019
MetaboAnalyst. The metabolite-metabolite network was constructed by
Metscape plug-in in Cytoscape software.
2.8. Western blot analysis
Electrophoresis gel was prepared using Biyuntian 10%SDS gel kit
(using 10 mm comb), and samples were prepared after gel solidification,
4 uL marker, 10 uL tissue sample. The electrophoresis conditions were as
follows: The concentrated glue voltage was 80 V and the time was 20
min until the protein and marker were flattened at the upper glue and
the lower glue. The separation glue voltage is 120 V and the time is 90
min. Stop when Marker distributes evenly and moves to the bottom of
gel. The PVDF membrane was clipped and put into methanol solution for
activation for 2–3 min, then the target protein gel was removed and
placed on the PVDF membrane, and the membrane was transferred for
90 min by cross-flow wet rotating method with 350 mA (adding ice
water to prevent the temperature from being too high). After the
membrane was transferred, the membrane was removed and put into 5%
skim milk, and closed in a shader at room temperature for 1 h. After
blocking, wash skim milk, use primary antibodies including TLR4
Antibody (1:1000, Affinity), Phospho-NF-kB p65 Antibody (1:1000,
Affinity), NF-kB p65 Antibody (1:1000, Affinity), Phospho-IKB alpha
Antibody (1:1000, Affinity), IKB Antibody (1:1000, Affinity) and beta
Actin Antibody (1:1000, Affinity) were incubated overnight at 4 ◦ C. The
next day, incubate the residual antibody on the TBST eluted membrane
for 3–5 times, 5 min each time, incubate with HRP-labeled Goat Anti-
rabbit IgG Antibody (1:2000, Zhongshan Jinqiao) after washing, and
incubate the residual antibody on the TBST eluted membrane for 2 h at
room temperature on a shaker. Incubate the antibody remaining on the
TBST eluted membrane 3–5 times, 5 min each time.
2.9. Statistical Analysis
Values represent the mean ± SD (standard deviation). Statistical
significance of differences was determined by one-way ANOVA with
Bonferroni’s post-hoc comparison, where appropriate. Thereby, P-
values less than 0.05 were considered significant. GraphPad Prism 8.0.2
software was used for statistical analysis.
3. Results
3.1. Regulatory effect of ginseng components on biological parameters in
rats with Qi-deficiency
3.1.1. Effect of different ginseng components on body weight and oxidative
stress
To determine the effect of ginseng extract on the body weight of rats
with Qi-deficiency, body weight was measured on days 0, 7, 14, 21, and
28 after administration (Fig. 1A). Over time, the body weight of rats in
the model group was significantly lower than that of rats in the control
group. The body weights of rats in all treatment groups were higher than
those in the model group, especially in the GE group. To evaluate the
regulatory effect of ginseng components on oxidative stress in Qi-
deficiency, the major oxidative stress indicators, MDA content and
SOD activity, were determined. As shown in Fig. 1, the MDA content in
the myocardium and thigh muscle was significantly higher in the model
group than in the control group. GE predominantly downregulated the
MDA content in the myocardium (P < 0.05) and thigh muscle (P < 0.01).
The MDA content in the GS and GP group exhibited downward trends;
however, only the MDA content in the GP group was significantly
decreased (P < 0.05). SOD activity was found to significantly decrease in
the myocardium and thigh muscles (P < 0.05) in response to Qi-
deficiency. GE administration significantly reduced SOD activity in the
myocardium (P < 0.05) and thigh muscles (P < 0.01) compared to that
obtained with model treatment. A considerable reduction in SOD ac­
tivity was also detected in the GS and GP groups compared to that in the
Y. Li et al. Journal of Pharmaceutical and Biomedical Analysis 242 (2024) 116019

Fig. 1. Regulatory effect of ginseng water extracts on body weight, energy metabolism and oxidative stress indicators in deficiency rats. The alteration of (A) body
weight in different groups. The serum activity of energy metabolism indicators (B) LDH, (C) ATPase, and (D) CK in different groups. The content of MDA, and the
activity of SOD in the myocardium (E, G) and thigh muscle (F, H) in different groups. Compared with the Con group, # P < 0.05, ## P < 0.01; Compared with the
Model group, * P < 0.05, ** P < 0.01.

model group; however, the difference was not significant. These results
indicate that GE have antioxidative effects in rats with Qi-deficiency,
which may contribute to their regulatory effects in Qi-deficiency.
3.1.2. Effect of different ginseng components on energy metabolism
Energy metabolism disorder is a major syndrome induced by Qi-
deficiency [20]. Hence, several energy metabolism indicators,
including LDH, ATPase, and CK, were assessed to evaluate the effects of
ginseng extract on rats with this deficiency. LDH is a key enzyme
involved in glycolysis and its level reflects the energy metabolism of an
organism [21]. ATPase and CK are involved in ATP generation, which
critically affects energy metabolism [22–24]. As shown in Fig. 1, muscle
LDH, ATPase, and CK activities in the model group significantly
decreased compared to those in the control group, indicating energy
metabolism disorders induced by Qi-deficiency. Treatment with GE and
GS clearly decreased LDH activity compared to model treatment
(P < 0.01). ATPase activity was downregulated in the GS group
compared to that in the model group (P < 0.05). The increased CK ac­
tivity in rats with Qi-deficiency was significantly decreased by GE, GS,
and GP. These results indicate that ginseng components can prominently
modulate energy metabolism disorders induced by Qi-deficiency, espe­
cially in the GE group.
3.1.3. Effect of different ginseng components on blood routine indexes
The spleen and thymus are important immune organs that are
extensively involved in immune responses. Therefore, the spleen and
thymus indices were evaluated to determine the immunomodulatory
effects of different ginseng components in Qi-deficiency. The spleen

4
Y. Li et al.
index (Fig. 2 A) and thymus index (Fig. 2 B) of rats in the model group
were significantly lower than those in the control group. In response to
the administration of the GE, the spleen index of the GE group signifi­
cantly increased compared to that in the model group (P < 0.05). The
thymus index of rats in the GP group (P < 0.01) and GS group (P < 0.05)
was upregulated compared to that in the Qi-deficiency group. The
thymus index was significantly decreased in rats with Qi-deficiency
(P < 0.01), which was markedly upregulated by GE administration
(P < 0.01). Other routine blood indexes were also detected, which could
predict the pathological changes in the body and serve as indicators in
the evaluation of Qi-deficiency syndrome [25], including white blood
cells (WBC), lymphocytes (LY), red blood cells (RBC), hemoglobin
(HGB), hematocrit (HCT), and platelets (PLT). As shown in Fig. 2, the
levels of RBC (P < 0.01), HGB (P < 0.01), HCT (P < 0.01), and PLT
(P < 0.01) were significantly decreased in rats with Qi-deficiency,
indicating that Qi-deficiency leads to disorders of hematological
indices in rats. After 30 days of treatment with GE, the levels of these
blood indices markedly increased, indicating that the GE could effec­
tively intervene in the symptoms of Qi-deficiency in rats. In particular,
the GE was found to exhibit an immunomodulatory effect in rats with
Qi-deficiency, with remarkable upregulation of immune cell numbers
and immune dysregulation-induced RBC dysregulation in rats adminis­
tered GE.
3.2. Regulatory effect of the ginseng components on the metabolic profile
of rats with Qi-deficiency
The srum metabolomic profiles of the positive and negative ion
patterns were obtained using UPLC-Q-TOF/MS. The raw data of serum
profiles were imported into EZinfo software (version 2.0) for multivar­
iate statistical analysis. In the PCA score diagram in Fig. 3, each dot
represents a serum sample, and samples from different groups are
clustered separately, indicating differences between the groups. Ac­
cording to the results shown in Fig. 3 in the positive ion mode (Fig. 3A),
the separation of the Con, Model, GE, GS, and GS groups was obvious. In
the negative ion mode (Fig. 3B), the PCA score plot showed better results
than that obtained in the positive ion model. The Qi-deficiency model
and control rats exhibited remarkable changes. The GE group partially
overlapped with the GS and GP groups, indicating that the metabolic
pathways involved in these three treatment groups also partially
Fig. 2. Effects of ginseng extract on tissue indexes and blood routine indexes in Qi-deficiency rats. The (A) spleen index, (B) thymus index, (C) WBC, (D) LY, (E) HGB,
(F) PLT, (G) HCT and (H) RBC in different groups. Compared with the Con group, # P < 0.05, ## P < 0.01; Compared with the Model group, * P < 0.05, ** P < 0.01.
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Journal of Pharmaceutical and Biomedical Analysis 242 (2024) 116019
overlapped. All three groups were similar to the Con group, indicating
that the metabolic profile altered by Qi-deficiency was partially restored
by treatment with the ginseng extract. QC samples are used to evaluate
system stability; QC samples were clustered together in both the positive
and negative ion modes of PCA, indicating good stability of the system.
To evaluate the therapeutic effect of the three drug administration
groups on Qi-deficiency syndrome, OPLS-DA plots were used to screen
for metabolic biomarkers between the model group and the GE, GS, and
GP groups in positive (Fig. 3 C-E) and negative (Fig. 3 F-H) ion modes.
The parameters of R2Y and Q2 were 99% and 96% in the model and GE
groups, 97% and 95% in the model and GS groups, and 98% and 96% in
the model and GP groups, respectively, in the positive ion mode. In the
negative ion mode, R2Y and Q2 were 98% and 94% in the model and GE
groups, 98% and 94% in the model and GS groups, and 97% and 93% in
the model and GP groups, respectively, indicating that the OPLS-DA
score plots have excellent stability and applicability. The red dots in
the S plots (Supplementary Fig. S5) represent compounds with VIP > 1.
Compounds with VIP > 1 and P < 0.05 were screened preliminarily, and
the reliability of the compounds was further verified by obtaining sec­
ondary fragment information of the compounds.
3.3. Identification of serum biomarkers for rats with Qi-deficiency
To accurately identify the biomarkers, we re-entered the candidate
compound information into the Progenesis QI, which was matched
against the actual molecular weight and fragment ions of the compound
in databases, including HMDB, METLIN, and ChemSpider. The error was
set to within 10 ppm. Disease-related compounds were screened, and
their structures were identified by comparison with standard substances
and mass spectrometry. The identified serum biomarkers for rats with
Qi-deficiency are shown in Table 1, with changing trends in the different
groups. A total of 27 metabolites were identified as serum metabolic
biomarkers in rats with Qi-deficiency. Among these metabolic bio­
markers, the relative intensity of 25 biomarkers altered by Qi-deficiency
was partially reversed by GE administration, and 18 biomarkers and 18
biomarkers were altered by GS and GP, respectively. Consistent with the
results of the PCA score plots, overlaps were found in the metabolic
biomarkers regulated in the GE, GS, and GP groups, including citric acid,
L-tryptophan, kynurenic acid, and oleic acid. Ginseng components have
been suggested to predominantly regulate the altered serum metabolic
Y. Li et al.
Fig. 3. Differential metabolic profile in response to Qi-deficiency and ginseng water extracts treatment. The PCA Score plots of the Con group (■), model group (■),
GE group (■), GS group (■), and GP group (■) in positive ion mode (A) and negative ion mode (B). OPLS-DA Score plots of between GE group, GS group, GS group
and model group in positive ion (C-E) and negative ion (F-H) mode, respectively.
profile in rats with Qi-deficiency, with GE administration exhibiting a
better regulatory effect than GS and GP administration.
A heatmap was generated to illustrate the change trends of metabolic
biomarkers identified between the model and control groups (Fig. 4A).
In the heatmap, metabolites with increased expression values are coded
from blue to red. Treatment with the ginseng extract partially reversed
the Qi-deficiency-induced alteration in these metabolites, which may
subsequently contribute to the protective effect of ginseng against Qi-
deficiency.
Metabolite-metabolite interactions were explored using Metab­
oAnalyst, which helped highlight the potential functional relationships
between the 27 metabolic biomarkers in rats with Qi-deficiency. As
shown in Fig. 4B, citric acid, oleic acid, L-tryptophan, glucose, and
galactose were highly ranked according to the degree value of the
network, suggesting that these biomarkers were affected by Qi-
deficiency. Notably, the serum levels of citric acid, oleic acid, L-tryp­
tophan, and galactose were partially reversed by GE, GS, and GP
administration, indicating that different ginseng components could
significantly modulate the biological alterations induced by Qi-
deficiency, especially GE and GS.
3.4. Pathway enrichment analysis of the effect of ginseng components on
Qi-deficiency
Pathway enrichment analyses were performed to explore the bio­
logical characteristics of the biomarkers affected by ginseng compo­
nents. As shown in Fig. 5, the serum biomarkers of Qi-deficiency were
significantly enriched in tryptophan metabolism, butanoate meta­
bolism, the citrate cycle, biosynthesis of unsaturated fatty acids, and
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Journal of Pharmaceutical and Biomedical Analysis 242 (2024) 116019
synthesis and degradation of ketone bodies. According to the pathway
enrichment results, the major pathways by which ginseng extract
affected Qi-deficiency were found to differ. In response to GE adminis­
tration, most of the Qi-deficiency-related biomarkers were regulated,
with similar pathways being enriched in the GE group (Fig. 5A). The
pathways that were significantly influenced by GS treatment were
butanoate metabolism, citrate cycle, biosynthesis of unsaturated fatty
acids, and synthesis and degradation of ketone bodies. For GP treatment,
primary bile acid biosynthesis, tryptophan metabolism, citrate cycle,
and biosynthesis of unsaturated fatty acids were the major pathways
enriched in Qi-deficiency. This result suggests that the GE group could
critically modulate most of the pathways affected by Qi-deficiency,
exhibiting a relatively better list of therapeutic effects than GS and GP
on Qi-deficiency in rats.
A correlation network based on the KEGG pathway database was
constructed to illustrate the potential links between the biomarkers
affected by ginseng extract (Fig. 6). Based on the results of serum
metabolomics, we determined the regulatory mechanism whereby the
ginseng extract ameliorates Qi-deficiency in rats. The related metabolic
pathways included unsaturated fatty acid metabolism, glycolysis, bile
acid biosynthesis, tryptophan metabolism, tricarboxylic acid (TCA)
cycle, ketone synthesis, and degradation. This network captured all
significant associations between metabolites, revealed a global corre­
lation structure and interconnections among different modules,
enhanced current understanding of the effect of GE on Qi-deficiency.
Y. Li et al. Journal of Pharmaceutical and Biomedical Analysis 242 (2024) 116019

Table 1
Identification and change trend of biomarkers.
Mode Identification RT (min) Measure mass (m/z) Change trend

Model/Cona GE/Modelb GS/Modelb GP/Modelb

## ** **
ESI+ 3-Hydroxyanthranilic acid 0.76 140.0684 0.29 2.64 2.35 1.02
Oxoglutaric acid 0.83 145.0143 0.36## 1.60** 1.18 * 1.04
D-Galactose 0.86 203.0526 0.21## 3.61** 2.52** 1.17 *
Citric acid 0.92 191.0198 4.67## 0.45** 0.57** 0.72 *
Acetoacetic acid 1.52 188.0706 5.26## 3.87** 1.21 * 1.02
L-Tryptophan 2.89 204.0898 0.28## 2.69** 2.65** 1.45**
4,6-Dihydroxyquinoline 3.76 162.0675 0.35## 1.69** 1.05 1.16 *
Retinoic acid 4.94 301.2166 3.85## 0.71 * 0.89 0.78 *
Tryptophanol 8.95 184.0734 0.27## 1.21 * 1.07 1.14 *
Tetracosahexaenoic acid 9.08 357.2778 4.93## 0.41** 0.62** 0.77 *
ESI- D-Glucose 0.54 225.0626 3.29## 0.76 * 0.80 * 0.89
2-Hydroxybutyric acid 0.89 103.0405 2.86## 0.74 * 0.82 * 0.92
Gluconic acid 1.87 241.0564 0.42## 1.47** 1.22 * 1.04
Ketoleucine 2.28 129.0559 0.54## 1.27 * 1.03 1.20 *
Kynurenic acid 2.76 162.0675 0.58## 2.10** 2.01** 1.23 *
Indolelactic acid 3.24 204.0672 0.42## 2.33** 1.15 * 1.10
Taurodeoxycholic acid 3.78 498.2882 6.79## 0.60** 0.72 * 0.96
Glycocholic acid 3.99 464.3015 0.31## 1.17 * 1.06 1.10 *
21-Deoxycortisol 4.02 391.2125 0.48## 1.15 * 1.13 * 1.14 *
Cholic acid 4.39 408.2873 3.26## 0.76 * 0.63** 0.75 *
Deoxycholic acid 4.51 392.2926 0.39## 1.47** 1.26 * 1.21 *
Mestranol 4.87 355.1866 0.77# 0.92 0.91 0.87
Chenodeoxycholic acid 5.02 437.2903 0.39## 1.98** 1.12 1.33 *
Arachidonic acid 5.19 303.2322 4.01## 1.20 * 1.13 * 1.16 *
Isodesmosine 5.80 571.2885 1.22# 0.99 0.95 0.97
Oleic acid 7.23 281.2477 0.42## 1.28 * 1.14 * 1.21 *
Valeric acid 7.31 147.0664 0.22## 1.93** 1.01 1.26 *
a
Compared with the Con group, # P < 0.05, ## P < 0.01.
b
Compared with the Model group, * P < 0.05, ** P < 0.01.

Fig. 4. Modulatory effect of ginseng on metabolic biomarkers of Qi-deficiency rats. (A) Heatmap showing the intensities of potential biomarkers in different groups.
(B) Metabolite-metabolite interaction network of Qi-deficiency metabolic biomarkers.

3.5. Metabolite-gene network analysis of the effect of the ginseng water
extracts on Qi-deficiency
To further elucidate the metabolite-related targets affected by Qi-
deficiency and ginseng water extracts, a network linking the metabolic
biomarkers and target genes was constructed using the Metscape plugin
in the Cytoscape software. Nodes with a connective degree ≥ 3 in the
metabolite-gene network were considered to have a significant contri­
bution to the metabolic process changes induced by Qi-deficiency. A
total of 111 genes. including GCG, CAT, PCK2, LDHA, IL6, IL8, NFKB1,
and NR4A1, were observed to be highly related to the metabolic bio­
markers in rats with Qi-deficiency (Fig. 7A). These genes and their

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Y. Li et al. Journal of Pharmaceutical and Biomedical Analysis 242 (2024) 116019

Fig. 5. Pathway enrichment analysis on the Qi-deficiency serum biomarkers affected by (A) GE, (B) GS, and (C) GP treatment.

Fig. 6. Correlation networks of potential biomarkers in response to therapeutic effects of ginseng water extracts on Qi-deficiency rats. Metabolites marked in red and
blue denote biomarkers up-regulated or down-regulated by GE (■), GS (■) and GP (■) administration.

encoded proteins are suggested to contribute to the progression of Qi-
deficiency in rats. The metabolite-gene networks of GE, GS, and GP
were also constructed (Fig. 7C-E), with 114, 80, and 97 targets,
respectively (Supplementary Fig. S6 and Supplementary Table S1). To
further illustrate the biological effects of ginseng water extracts on Qi-
deficiency, pathway enrichment analysis was performed for the target
genes affected by ginseng water extracts. The top 10 enriched pathways
in the GE, GS, and GP groups are presented in Fig. 7. The Qi-deficiency-
related targets were critically enriched in eicosanoid metabolism, nu­
clear receptors in lipid metabolism and toxicity, role of EGF receptor
transactivation by GPCRs in cardiac hypertrophy, mechanism of gene
regulation by peroxisome proliferators via PPARa, and toll-like receptor
pathway. Similar enriched pathways were observed with GE and GP for
Qi-deficiency. The common pathways affected by GE, GS, and GP in Qi-
deficiency were eicosanoid metabolism, the role of EGF receptor trans­
activation by GPCRs in cardiac hypertrophy, the Toll-like receptor
pathway, corticosteroids, and cardioprotection. NFKB1, FOS, EGF,
EDN1, PPARA, MAPK8, GNAS, and ANXA1 are genes involved in these
common pathways and are crucial targets for ginseng water extracts.
Hence, these results provide pathway and target information for the
therapeutic mechanisms of ginseng water extracts in Qi-deficiency.
3.6. Effect of different ginseng components on TLR4/NF-κB mediated
inflammatory response in rats with Qi-deficiency
Based on the metabolic gene network and pathway enrichment
analysis, the key biological processes involved in the regulation of Qi-
deficiency in the ginseng extract were identified as Toll like receptor
(TLR) signaling and myocardial function-related pathways, which are
closely associated with the inflammatory response. In addition, multiple
markers are involved in the regulation of muscle contraction coupling
and mitochondrial function. Therefore, to further determine the anti-
inflammatory effect of the ginseng extract on Qi-deficiency syndrome,
the activation of the TLR4/NF-κB signaling pathway and the levels of
inflammatory cytokines were determined in the different groups. As
shown in Fig. 8A-D, TLR4 expression was significantly increased in the
myocardium of rats with Qi-deficiency; however, this expression was
significantly downregulated after the administration of GE and GS.
Similarly, the expression of TLR4 in the thigh muscles of rats with Qi-
deficiency also significantly increased, displaying a trend similar to

8
Y. Li et al. Journal of Pharmaceutical and Biomedical Analysis 242 (2024) 116019

Fig. 7. Metabolite-gene network and target enrichment analysis of the intervention effect of different ginseng water extracts on Qi-deficiency rats. (A) The
metabolite-gene network of Qi-deficiency serum metabolic biomarkers. (B) Pathway enrichment of the targets genes affected by (B) Qi-deficiency, (C) GE, (D) GS and
(E) GP.

Fig. 8. Effect of ginseng components on myocardial TLR4/NF-κB pathway and pro-inflammatory factors in Qi-deficiency rats. (A-D) Western blot analysis of the
relative protein expression of TLR4, P-P65/P65 ratio and P-IκB/IκB ratio in different groups. (E-F) Serum pro-inflammatory factors levels of (E) TNF-α, (F) IL-1β, (G)
IL-6 and (H) INF-γ in different groups. Compared with the Con group, # P < 0.05, ## P < 0.01; Compared with the Model group, * P < 0.05, ** P < 0.01.

that of the myocardium. Moreover, the expression decreased to varying
degrees in each administration group, although the differences were not
statistically significant.
The levels of P-P65/P65 and P-IκB/IκB ratios, which were upregu­
lated in the myocardium of rats with Qi-deficiency, were downregulated
by GE treatment. These findings suggest that different ginseng compo­
nents, particularly GE, primarily inhibit the activation of the TLR4/NF-
κB signaling pathway induced by Qi-deficiency.
We proceeded to determine the serum levels of inflammatory cyto­
kines (Fig. 8E-H). TNF-α, IL-1β, and IL-6 are important inflammatory

9
Y. Li et al.
cytokines that participate in the body’s inflammatory response [26–28].
INF-γ plays an important role in the immune regulation of the body and
is one of the important immune regulators [29,30]. The model group
exhibited elevated levels of TNF-α and IL-1β, which were significantly
reduced by GE administration. Qi-deficiency resulted in increased IL-6
levels; however, no significant changes were observed following treat­
ment with the ginseng extract. However, the decreased serum level of
INF-γ in the model group was found to be upregulated in the GE group.
These findings suggest that ginseng water extracts can effectively
regulate inflammation induced by Qi-deficiency potentially by inhibit­
ing the TLR4/NF-κB signaling pathway.
4. Discussion
In this study, we established a Qi-deficiency model based on exper­
imental methods described by Lin et al. [3] and successfully verified its
effectiveness. In addition, we determined the optimal dose of GE by
administering different doses to the animals. Once the model and dosage
were established, we used a combination of metabolomics and phar­
macological research methods to evaluate the protective effects of
ginseng extract in rats with Qi-deficiency. A total of 27 serum metabolic
biomarkers were found to be primarily regulated by ginseng compo­
nents, particularly GE and GS, in rats with Qi-deficiency. These bio­
markers were found to be involved in energy metabolism (TCA cycle,
glycolysis/gluconeogenesis, and the synthesis and degradation of ketone
bodies), bile acid biosynthesis, unsaturated fatty acid metabolism, and
tryptophan metabolism. Metabolite network analysis revealed that TLR
signaling and myocardial function-related pathways are crucial biolog­
ical processes that are modulated by the ginseng extract against
Qi-deficiency. Further validation revealed that GE could significantly
reduce the inflammatory response and activation of the myocardial
TLR4/NF-κB pathway induced by Qi-deficiency. These findings suggest
that ginseng can prevent the progression of Qi-deficiency-related
dysfunction through metabolic management and modulation of in­
flammatory responses.
Qi-deficiency, identified as a deficiency of vital energy in traditional
Chinese medicine, manifests as chronic fatigue, dizziness, abdominal
distension, and other phenotypes [31,32]. Remarkable alterations in
energy metabolism are considered major contributors to Qi-deficiency
[20]. Consistently, an overall decrease in the biological indicators of
energy metabolism, including LDH, CK, and ATPase, was found in rats
with Qi-deficiency, indicating an inadequate energy supply after
long-term exhaustion. LDH is a key enzyme involved in glycolysis and its
levels reflect the energy metabolism of an organism [21]. ATPase and CK
are involved in ATP generation, which critically affects energy meta­
bolism [22–24]. However, GE and GP administration notably upregu­
lated the activity of LDH and CK, with GE exhibiting a relatively more
efficient performance than GS. GE was preliminarily demonstrated to
effectively interfere with disturbed energy metabolism in Qi-deficiency,
which was further validated via metabolomic analysis.
In the current study, dysregulated energy metabolism was also
observed in rats with Qi-deficiency via metabolomic analysis. The major
metabolites in the TCA cycle, glycolysis/gluconeogenesis, and the syn­
thesis and degradation of ketone bodies were markedly altered in the
serum of rats with Qi-deficiency. Glucose, gluconic acid, and LDH are
the major components of glycolysis during cellular respiration. Alter­
ations in metabolite content and enzyme activity could directly reflect
perturbed glycolytic metabolism during Qi-deficiency. Increased serum
glucose levels have been observed in the serum of rats with Qi-
deficiency, which could lead to organ damage. Similar alterations
have been reported in rats with Qi-deficiency after training, confirming
fatigue in swimmers [33,34]. The consumption of liver and muscle
glycogen after long-term exhaustion may be attributed to the elevated
levels of serum glucose. LDH activity is closely related to the level of
lactate, the end product of glycolysis under anaerobic conditions.
Decreased serum activity of LDH mediates the accumulation of lactate
10
Journal of Pharmaceutical and Biomedical Analysis 242 (2024) 116019
and is considered an indicator of inhibited glycolytic metabolism in rats
with Qi-deficiency [35]. In contrast, increased LDH activity accelerates
in vivo removal of lactate under anaerobic conditions, thereby allevi­
ating fatigue [36]. In the current study, reduced serum glucose content
and serum lactate dehydrogenase activity in response to treatment with
the ginseng extract revealed a modulatory effect against glycolysis
metabolism dysregulation induced by Qi-deficiency.
The TCA cycle is one of the most important energy metabolic pro­
cesses and a downstream metabolic pathway of glycolysis. In fact, this
cycle is the most efficient pathway for in vivo energy production and
reflects the function of mitochondrial energy metabolism [37]. Citric
acid and oxoglutaric acid, two substrates of rate-limiting enzymes in the
TCA cycle, were markedly altered and identified as metabolic bio­
markers of Qi-deficiency via metabolomics. Consistent with the current
results, decreased oxoglutaric acid content was observed in chronic fa­
tigue conditions, indicating inhibition of the TCA cycle in response to
Qi-deficiency. However, the serum citric acid content of rats with
Qi-deficiency significantly increased. Citric acid is produced in the
mitochondria and used in the TCA cycle or released into the cytoplasm
through a mitochondrial carrier. Cytosolic citric acid plays a regulatory
role in the production of inflammation intermediates, including arach­
idonic acid and NO [38,39]. The overall inhibition of the TCA cycle and
the increase in citrate under inflammatory conditions have been
revealed previously [40]. The disordered TCA cycle observed in rats
with Qi-deficiency indicates mitochondrial dysfunction and an inflam­
matory response. However, treatment with ginseng extract, especially
GE, can partially reverse this disturbance. These results indicate that
ginseng extract could effectively regulate the energy metabolism
disturbance that occurs in rats with Qi-deficiency, thereby critically
contributing to its modulatory effect on mitochondrial function and
inflammatory responses.
The altered tryptophan metabolic pathway in rats with Qi-deficiency
is modulated by ginseng components, primarily GE. Tryptophan is an
essential amino acid that plays a fundamental role in physiology and
biochemistry [41]. L-tryptophan, 3-hydroxyanthranilic acid, and 4,
6-dihydroxyquinoline, are metabolites in tryptophan metabolism [42].
Changes in these amino acid concentrations indicate a shift in the bal­
ance between nutrient intake and consumption [43]. According to the
results of this study, the contents of L-tryptophan, 3-hydroxyanthranilic
acid, and 4,6-dihydroxyquinoline in the Qi-deficiency group signifi­
cantly decreased, and the three metabolites exhibited different upre­
gulation trends after treatment with each active ingredient of ginseng.
The levels of L-tryptophan and 3-hydroxyanthranilic acid in the GS
group and 4, 6-dihydroxyquinoline in the GP group increased. Such
finding indicates that ginseng has a good regulatory effect on the im­
mune system of rats with Qi-deficiency. The active components of
ginseng exhibit different regulatory effects, with GE exerting the most
significant effect.
Several biomarkers in rats with deficiency are involved in the
biosynthesis of unsaturated fatty acids, including oleic acid and arach­
idonic acid, which can be primarily modulated by ginseng extract
administration. Fatty acids are essential components of cellular lipid
metabolism and are involved in intracellular signal transduction in
several physiological and pathological processes [44]. Decreased serum
oleic acid content was observed in rats with Qi-deficiency; however, this
decrease was reversed by treatment with the ginseng extract, particu­
larly GE. A similar change trend in oleic acid levels was found in in­
dividuals with chronic fatigue compared to age-matched controls [45].
Oleic acid, an omega-9 unsaturated fatty acid, is implicated in the
regulation of fatty acid oxidation, anti-inflammation, and improvement
of insulin sensitivity [46]. Oleic acid can attenuate triglyceride accu­
mulation and mitochondrial β-oxidation in skeletal muscle cells, and
subsequently prevent activation of the NF-κB related impairment of in­
sulin signaling and inflammation [46]. In skeletal muscle cells, oleic acid
can increase mitochondrial mass and maximum respiration, which
contribute to high oxidative capacity and resistance to fatigue [47]. The
Y. Li et al.
upregulated serum levels of oleic acid in the GE group indicated that the
ginseng extract promoted exercise performance and fatigue resistance in
rats with Qi-deficiency. Arachidonic acid, an omega-6 polyunsaturated
fatty acid, is also a biomarker for Qi-deficiency and serves as a classical
second messenger that is critically involved in impaired insulin action
and inflammatory responses in the skeletal muscle [48]. Arachidonic
acid can effectively inhibit the palmitic acid-induced inflammatory re­
sponses and insulin resistance in C2C12 myotubes, primarily by modu­
lating the activation of the NF-κB-dependent pathway [10,49]. The
content of arachidonic acid in the serum of rats in the Qi-deficiency
group was significantly lower than that in rats in the control group,
which was significantly reversed by treatment with GE, GS, and GP. The
total ginseng extract, ginsenoside, and ginseng polysaccharide can pre­
vent the progression of inflammation response and insulin resistance in
Qi-deficiency, with mechanisms related to the NF-κB-dependent
pathway.
Based on pathway enrichment and network analysis, ginseng water
extracts may prevent the progression of Qi-deficiency mainly by
modulating inflammatory response-related process, especially the TLR/
NF-κB signaling pathway. Hence, we further validated the regulatory
effect of different ginseng components on the TLR4/NF-κB signaling
pathway by evaluating the protein expression of TLR4 and phosphory­
lation of IκB and P65 in the myocardium and skeletal muscle of different
groups. Qi-deficiency was found to induce overexpression of TLR4 and
phosphorylation of IκB and p65 in the myocardium and skeletal muscle.
Activation of the TLR4 complex, a pattern recognition receptor, is crit­
ically involved in chronic inflammation and oxidative stress in cardio­
vascular disorders [50]. NF-κB is the major downstream target of TLR4,
a central mediator of inflammatory processes, that play a key role in
innate and adaptive immune responses [51]. According to recent
studies, myocardial inflammation is maintained by TLR4 and its
downstream phosphorylation of IκB-α in the NF-κB pathway, and in­
duces the expression of pro-inflammatory factors, such as IL-1 and TNFα
[52–54]. Prior studies have shown that long-term exhaustion may lead
to cardiac hypertrophy, myocardial inflammation, and pathological
cardiac remodeling [55,56]. However, limited studies have revealed the
connection between Qi-deficiency and myocardial dysfunction.
In the present study, by integrating metabolomics-based bio­
informatic analysis with biological validation, we obtained tentative
initial evidence that Qi-deficiency may lead to myocardial inflammation
by activating the TLR4/NF-κB signaling pathway. Treatment with the
ginseng extract, especially GE, substantially suppressed the over­
expression of the TLR4 pathway in the myocardium and modulated the
overall inflammatory cytokines induced in rats with Qi-deficiency.
These results highlight the potential cardioprotective and anti-
inflammatory effects of ginseng water extracts on Qi-deficiency; how­
ever, further systematic investigation and verification are needed.
5. Conclusions
In the present study, the protective effects and modulatory mecha­
nisms of ginseng water extract, ginsenosides, and ginseng poly­
saccharides in rats with Qi-deficiency were systematically investigated
using metabolomics-based bioinformatic analysis and biological vali­
dation. Treatment with the ginseng extract was found to effectively
restore the energy metabolic dysfunction caused by Qi-deficiency by
regulating the TCA cycle, glycolysis, and biosynthesis of unsaturated
fatty acids, especially treatment with GE. GE was found to exhibit car­
dioprotective and anti-inflammatory effects in Qi-deficiency primarily
by inhibiting the TLR4/NF-κB signaling pathway. Moreover, the GE had
a better effect at reversing Qi-deficiency than ginsenosides and ginseng
polysaccharides. These findings indicate that the ginseng water extract
can serve as an effective preventive measure against the progression of
Qi-deficiency by regulating metabolism and inflammatory responses.
11
Journal of Pharmaceutical and Biomedical Analysis 242 (2024) 116019
Funding
This research was supported by the National Natural Science Foun­
dation of China U19A2012.
Ethical statement
All the animal studies were performed according to institutional
guidelines for the care and use of laboratory animals. Protocols were
approved by the Laboratory Animal Ethics & Welfare Committee in
College of Basic Medical Sciences of Jilin University (Approved No.
2021015).
CRediT authorship contribution statement
Li Yanyi: Writing – review & editing, Writing – original draft,
Validation, Methodology, Formal analysis, Conceptualization. Men
Lihui: Writing – review & editing, Validation, Supervision, Data cura­
tion. Liu Zhongying: Writing – review & editing, Resources, Data
curation. Wang Meiyuan: Software, Methodology, Formal analysis. Li
Hanlin: Investigation, Formal analysis. Wu Yi: Writing – review &
editing, Supervision, Methodology. Liu Zhiqiang: Supervision, Project
administration, Funding acquisition. Liu Shu: Validation, Supervision,
Project administration. Pei Shuhua: Validation, Software, Investiga­
tion. Gao Yang: Validation, Methodology, Investigation, Formal
analysis.
Declaration of Competing Interest
All authors of the paper titled “UPLC-QTOF-MS Based Metabolomics
Unravel the Modulatory Effect of Active Components from Ginseng on
Qi-deficiency Rats”declare no conflicts of interest.
Data Availability
Data will be made available on request.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.jpba.2024.116019.
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