Journal of Chromatography B 1226 (2023) 123676

Contents lists available at ScienceDirect

Journal of Chromatography B
journal homepage: www.elsevier.com/locate/jchromb

Systems biology strategy through integrating metabolomics and network

pharmacology to reveal the mechanisms of Xiaopi Hewei Capsule improves
functional dyspepsia
Runhua Liu a, b, 1, Tianyi Li a, 1, Haoran Xu a, Gengyuan Yu a, Tonghua Zhang a, Jiaqi Wang a,
Yu Sun a, Yuelin Bi a, Xin Feng a, Hao Wu a, Chenning Zhang a, c, *, Yikun Sun a, *
a
School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, China
b
Department of Pharmacy, Children’s Hospital Affiliated to Capital Institute of Pediatrics, Beijing 100020, China
c
Department of Pharmacy, Zigong First People’s Hospital, Zigong, China

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

Keywords: Functional dyspepsia (FD) is one of the more common functional disorders, with a prevalence of 20–25 %. It
Xiaopi Hewei Capsule seriously affects the quality life of patients. Xiaopi Hewei Capsule (XPHC) is a classic formula originated from the
Functional dyspepsia Chinese Miao minority. Clinical studies have demonstrated that XPHC can effectively alleviate the symptoms of
Metabolomics
FD, but the molecular mechanism has not been elucidated. The purpose of this work is to investigate the
Network pharmacology
mechanism of XPHC on FD by integrating metabolomics and network pharmacology. The mice models of FD
Gene-metabolite interactions
were established, and gastric emptying rate, small intestine propulsion rate, serum level of motilin and gastrin
were evaluate to study the interventional effect of XPHC on FD. Next, a metabolomics strategy has been
developed to screen differential metabolites and related metabolic pathways induced by XPHC. Then, prediction
of active compounds, targets and pathways of XPHC in treating FD were carried out by commonly used network
pharmacological method. Finally, two parts of the results were integrated to investigate therapeutic mechanism
of XPHC on FD, which were preliminary validated based on molecular docking. Thus, twenty representative
different metabolites and thirteen related pathways of XPHC in treating FD were identified. Most of these me­
tabolites were restored using modulation after XPHC treatment. The results of the network pharmacology
analysis showed ten crucial compounds and nine hub genes related to the treatment of FD with XPHC. The
further integrated analysis focused on four key targets, such as albumin (ALB), epidermal growth factor receptor
(EGFR), tumor necrosis factor (TNF) and roto-oncogene tyrosine-protein kinase Src (SRC), and three represen­
tative biomarkers such as citric acid, L-leucine and eicosapentaenoic acid. Furthermore, molecular docking re­
sults showed that ten bioactive compounds from XPHC have good binding interactions with the four key genes.
The functional enrichment analysis indicated that the potential mechanism of XPHC in treating FD was mainly
associated with energy metabolism, amino acid metabolism, lipid metabolism, inflammatory reactions and
mucosal repair. Our work confirms that network pharmacology-integrated metabolomics strategy is a powerful
means to reveal the therapeutic mechanisms of XPHC improves FD, which contribute its further scientific
research.

Abbreviations: FD, functional dyspepsia; PDS, postprandial distress syndrome; EPS, epigastric pain syndrome; Hp, helicobacter pylori; TCM, traditional Chinese
medicine; CA, Cynanchum auriculatum Royle ex Wight; LRT, leaves of Rosa roxburghii Tratt.; SBL, Salix babylonica L.; PN, Panax notoginseng (Burkill) F.H.Chen.; TNF-α,
tumor necrosis factor-α; MTL, motilin; GAS, gastrin; IA, iodoacetamide; ESI, electrospray ionization source; ELISA, enzyme linked immunosorbent assay; QC, quality
control; PCA, principal component analysis; OPLS-DA, orthogonal partial least squares discriminant analysis; VIP, value of variable importance in the projection;
HMDB, Human Metabolome database; BPI, basic peak ion; ACTH, plasma adrenocorticotrophic hormone.
* Corresponding authors at: School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, China (C.Z.).
E-mail addresses: [email protected] (C. Zhang), [email protected] (Y. Sun).
1
These authors have contributed equally to this work and share first authorship.

https://doi.org/10.1016/j.jchromb.2023.123676
Received 26 November 2022; Received in revised form 5 March 2023; Accepted 13 March 2023
Available online 14 April 2023
1570-0232/© 2023 Published by Elsevier B.V.
R. Liu et al. Journal of Chromatography B 1226 (2023) 123676

1. Introduction regulate gastrointestinal hormone content, and relieve gastrointestinal

motility disorders [31]. Therefore, we have reason to believe that XPHC
Undoubtedly, health has a profound impact on people’s daily lives, has potential and widely application in treating FD. However, the un­
and encourages motivation, confidence, vitality and social interactions. determined mechanism of XPHC in treating FD makes many patients
However, it is inevitable that illness will always come, and bring pain worry about this drug. Thus, our work contributes to promoting the
and anxiety. Functional dyspepsia (FD), as a digestive system disease acceptance and application of XPHC.
with a global incidence up to 40 %, continues to endanger people’s The multi-component and multi-target features of TCM make eluci­
physical and mental health [1,2]. According to the latest Rome IV dation of the interventional mechanism of TCM difficult. Based on the
consensus, FD is divided into three subgroups based on symptoms: (1) whole level of metabolites, metabolomics is regarded as a key technol­
postprandial distress syndrome (PDS) with post-prandial fullness or ogy to investigate the pathogenesis of diseases and the interventional
early satiation; (2) epigastric pain syndrome (EPS) with a sensation of mechanism of drugs [32]. However, traditional metabolomics only fo­
pain or burning in the epigastrium; (3) PDS-EPS overlap syndrome [3,4]. cuses on the terminal changes of metabolites, which cannot illuminate
The diverse clinical symptoms of FD are closely correlated with the the mechanism of metabolite changes. Moreover, the upstream path­
complexity of pathogenic factors [5]. Disrupted gastrointestinal ways and proteins that regulate metabolites are poorly understood [33].
motility, visceral hypersensitivity, psychological illness, unbalanced Therefore, metabolomics alone may have some limitations. In the
diet, intestinal inflammation and Helicobacter pylori (Hp) infection context of systems biology, network pharmacology visualises the con­
have been reported in FD [6,7]. To alleviate the harm caused by this nections of different systems by using multivariate databases and pro­
disease, scientists are tirelessly engaged in research aiming to investi­ vides a new perspective for researching the therapeutic mechanisms of
gating the pathogenic mechanism of FD, developing new drug formu­ medicine [34]. Regrettably, the implementation of network pharma­
lations and exploring pharmacological interventions. cology relies on a series of public databases, and only shows the
In recent years, there has been increased interest in using traditional component-target-pathway connection [35]. The mechanism obtained
Chinese medicine (TCM) for FD treatment, as it has significant efficacy via network pharmacology still needs to be verified [36].
and few side-effects [6–8]. The discovery of new TCM formulas and Researchers point out that the combination of metabolomics and
exploring the mechanisms of interventions have always been a vibrant network pharmacology could make up for their respective shortcomings
field of research for natural medicine scientists worldwide. Xiaopi to clarify the interventional effect of drugs on disease [37,38]. For
Hewei Capsule (XPHC), which originated from the classical folk ‘Miao example, the mechanisms of hydroxysafflor yellow A against acute
minority’ medicine, has an impressive clinical experience in treating traumatic brain injury have been revealed by using metabolomics and
vomiting, fullness or distension or pain in the stomach [9,10]. In recent network pharmacology [37]. Shu-Yue Qu et al. have analysed the anti-
years, researchers have also begun to focus on the in-depth study of depressant activity of Huang-Lian Jie-Du Decoction based on network
XPHC with the government’s emphasis on ethnic medicine [10,11]. pharmacology and metabolomics [33]. Network pharmacology and
XPHC is composed of Cynanchum auriculatum Royle ex Wight ([Apoc­ metabolomics also played a crucial role in uncovering the mechanism by
ynaceae], CA), leaves of Rosa roxburghii Tratt. ([Rosaceae], LRT), Salix which Shengqi fuzheng injection regulates mitochondrial dysfunction in
babylonica L. ([Salicaceae], SBL) and Panax notoginseng (Burkill) F.H. cancer-related fatigue [39]. Thus, the potential mechanism of XPHC in
Chen ([Araliaceae], PN) in a proportion of 6:4:3:3 [9]. Compounds in CA treating FD may also be revealed through metabolomics and network
include C21-steroids, acetophenones, terpenoids and alkaloids [12]. pharmacology.
Several researchers also showed that CA and its components like cyn­ Complementarily, improvements in LC-MS technology have brought
andione A, wilfoside K1N or C21-steroids compounds have effects of much surprises to scientific researchers, which have been widely used in
anti-inflammation[13,14], liver-protection [15], anti-oxidation [16], research involving metabolomics and network pharmacology [40]. MS
anti-drepression and so on [17]. CA was approved as functional food technology makes it possible to discover compound changes among
(food supplement) in China. Compounds in LRT were amino acids, fla­ different samples during the process of metabolomics. The confirmation
vonoids, organic acids and triterpenes, which possess anti-oxidation, of the active compounds of TCM via MS technology prevents compounds
hypoglycemic, heart protection and anti-inflammation activity from being missed due to parameter limitations [41].
[18,19]. The phenolic compounds contained in LRT showed excellent Molecular docking technology is a widely used to investigating the
performances on antioxidant activities and α-glucosidase inhibitory relationship between drugs and targets, and is suitable for exploring the
potency [20]. The flavonoids in SBL are exhibited significant antioxi­ mechanism of TCM in treating diseases [42]. In this study, the inter­
dant activities [21], which is also associated with its metabolites ventional effect, metabolites and pathway of XPHC in treating FD were
[22,23]. Panax notoginseng saponins (PNS) are the most abundant and determined through non-targeted metabolomics based on UHPLC-Q-
representative compounds of PN [24]. Clinical studies have demon­ Exactive Orbitrap/MS technology. Subsequently, the component-
strated that XPHC can effectively alleviate the symptoms of patients target-pathway connection was established via network pharma­
with FD by shortening the emptying time of the stomach, thus relieving cology. Topological analysis was used to screen key target genes. A gene-
upper abdominal pain and bloating [11,25]. metabolite interaction network was constructed to explore the interac­
Although there are few existing studies on XPHC in treating FD, re­ tion between key genes and metabolites, thus contributing to under­
searches on four herbs in the prescription are nothing new. CA is an standing the mechanisms of XPHC in treating FD. The research flowchart
important herbal drug for the treatment of gastric disorders (indigestion was shown in Fig. 1.
and stomachache) in the ethnomedicine of the Tujia and Hmong/Miao
[26]. Extracts of CA can regulate the levels of tumour necrosis factor-α 2. Materials and methods
(TNF-α), motilin (MTL) and gastrin (GAS) in FD rats, and thereby pro­
mote gastrointestinal motility and improve gastrointestinal function 2.1. Plant material and reagents
[27,28]. LRT have the effect of nourishing the stomach and eliminating
food, and is traced back to Chinese Materia Medica (Miao Medicine Xiaopi Hewei capsules, Cynanchum auriculatum Royle ex Wight,
Volume) [19]. Salicin, the main compounds of SBL, can transform into leaves of Rosa roxburghii Tratt., Salix babylonica L. and Panax notoginseng
salicylic acid after interacting with enzymes in vivo [29]. Salicylic acid (Burkill) F.H.Chen were provided and quality controlled by Guiyang
is anti-inflammatory and suppresses pain, which can relieve the burning Yongle Pharmaceutical Co. Ltd. (Guizhou, China). The herbs were
sensation in the stomach [23]. PN promotes the recovery of Hp-infected identified by professor Yikun Sun (Beijing University of Chinese Medi­
mice by reducing gastric secretion and resisting free radical damage cine, Beijing, China). Domperidone used in this work was from Xian
[30]. PNS can significantly improve gastrointestinal motility in FD rats, Janssen Pharmaceutical Ltd. (Xian, China). Iodoacetamide (IA) was

2
R. Liu et al. Journal of Chromatography B 1226 (2023) 123676

Fig. 1. The research flowchart.

purchased from Sigma-Aldrich (Shanghai, China). Sucrose was pur­ Use of Laboratory Animals published by the National Institutes of Health
chased from Beijing Biodee Biotechnology Co., Ltd. (Beijing, China). (NIH Publications No. 85-23, revised 1996).
Acetonitrile, formic acid and methanol were used in the current work Thirty-five mice were randomly allocated into 5 groups (n = 7 per
(all LC-MS grade) and purchased from Thermo Fisher Scientific (China) group): (1) the control group (CC); (2) the model group (FD); (3) the
Co. Ltd. Ultrapure water was obtained from a Milli-Q water purification model plus high-dose administration group (FD + XPHC-H); (4) the
system (Millipore, Bedford, USA). model plus low-dose administration group (FD + XPHC-L); (5) the
model plus domperidone administration group (FD + Dom). Iodoace­
2.2. Preparation of the decoction extraction tamide (IA) treatment, tail-clamping and alternate-day fasting were
imposed on mice to construct a functional dyspepsia model. The detailed
5 g powder of XPHC were decocted with ten times water for two process of the animal experiment was as follows. Mice in the CC group
hours (w/v). Meanwhile, 5 g CA, 5 g LRT, 5 g SBL and 5 g PN were were orally administrated 2 % sucrose solution in the morning with free
weighed from the same batch of crude herbs, then decocted using the access to chow. Mice in the model groups (FD; FD + XPHC-H;
same approach respectively. And the above sample is used for chemical FD + XPHC-L; FD + Dom) were treated with 0.1 % IA in 2 % sucrose
composition analysis. Sample processing is as follows: the decoction solution in the morning, stimulated by tail clamping (4 times a day for
liquid was centrifuged for 15 min with 12,000 r/min, and diluted to 5 30 min per cycle), and subjected to alternate-day fasting. Medication
times, take 5 uL of diluent for mass spectrometry analysis. In addition, was administered in the afternoon: (1) CC group (saline, 8 mL/kg, per
six times the amount of XPHC powder (30 g) was prepared in parallel by day); (2) FD group (saline, 8 mL/kg, per day); (3) FD + XPHC-H group
the same method, and filtered and concentrated to 0.225 g/mL (XPHC- (XPHC-H, 2.25 g/kg, per day); (4) FD + XPHC-L group (XPHC-L, 0.75 g/
H) and 0.075 g/mL (XPHC-L) for the animal experiment, respectively. kg, per day). (5) FD + Dom group (domperidone, 3 mg/kg, per day). The
high dose of XPHC administered to mice was converted from the three
times dosage administered to humans. The low dose of XPHC adminis­
2.3. Animal experiment and drug administration tered to mice was converted from the dosage administered to humans.
The mice received saline, XPHC, or domperidone by oral gavage for
Thirty-five specific pathogen-free male ICR mice (25–28 g) and six 1 weeks. Moreover, modelling and drug delivery were carried out at the
male specific-pathogen-free Sprague Dawley rats (180–250 g) were same time. A diagram of the model was shown in Fig. 2.
obtained from a commercial animal breeder (Beijing Sibeifu Biotech­ Furthermore, six male Sprague Dawley rats were orally adminis­
nology Co., Ltd. of China) with the license number ‘SCXK (Jing) 2019- trated XPHC-H decoction (2.25 g/kg) for one week, which provide
0010’. They were housed in an environmentally controlled room compounds information of XPHC in vivo.
maintained at 22 ± 2 ◦ C and 60 % ± 5 % relative humidity on a 12/12 h
light/dark cycle. They were given free access to a commercial standard
chow diet (Beijing Sibeifu Biotechnology Co., Ltd.; Beijing, China) and 2.4. Gastric emptying rate and small intestine propulsion rate
tap water, and allowed to adjust to their environment for 7 days before
experimentation was initiated. Animal experiments were approved by The mice were fasted for 12 h at the end of the trial having free access
the Animal Care and Use Committee of Beijing University of Chinese to water, and then orally administrated 5 % graphite carbon powder
Medicine and were performed according to the Guide for the Care and (0.4 mL) containing milk and glucose (WA1). 30 min later, blood was

3
R. Liu et al. Journal of Chromatography B 1226 (2023) 123676

Fig. 2. The diagram of functional dyspepsia model construction.

sampled from the eyes of the mice, and the serum was isolated by The separated supernatant was dried with nitrogen at 37 ◦ C and 100 µL
centrifugation at 3500 rpm (15 min at 4 ◦ C). The supernatants were methanol was added for redissolution, before centrifugation at
stored at − 80 ◦ C until further analysis. Subsequently, the mice were 12,000 rpm (10 min at 4 ◦ C). A 5 µL sample was then taken for analysis
culled, and the stomach was carefully excised and cleaned, recording the [42].
total weight (WA2) and the empty weight (WA3) respectively. The
percentage of gastric emptying was calculated according to the formula: 2.7.2. UPLC-QE-Orbitrap-MS instruments and conditions
Gastric emptying % = (1 − (WA2 − WA3)/WA1) × 100 %. The data was collected based on the UPLC-QE-Orbitrap-MS system
The small intestine was carefully removed. The overall length of the with a Waters HSS T3 UPLC C18 column (1.7 µm, 2.1 × 150 mm, Mil­
small intestine (from stomach to ileocecal junction) and the length of the ford, MA, USA). Chromatographic conditions were bettered by gradient
movement of graphite carbon powder were measured [43]. Small in­ elution technique with 0.1 % formic acid-aqueous solution (A) and
testine propulsion rate = (length of the movement of graphite carbon acetonitrile (B): 0 min-2 min: 1 % B; 2 min-6 min: 1 % B – 50 % B; 6 min-
powder/overall length of the small intestine) × 100 %. 15 min: 50 % B- 99 % B; 15 min-17 min: 99 % B; 17 min-19 min: 99 % B –
1 % B; 19 min-20 min: 1 % B. The flow rate was 0.3 mL/min, and the
2.5. ELISA assay temperature was 40 ◦ C. The injection volume was 5 μL. The sample
chamber temperature of the autosampler was 4 ◦ C. Data was acquired
Motilin (MTL) is a brain-gut peptide that is closely related to gastric via Xcalibur software (Thermo Fisher Scientific Corp, New York, MA,
motility and gastric emptying. Gastrin (GAS) distribution in the gastric USA).
antrum can stimulate gastric acid secretion and enhance gastric antral The ion source was an electrospray ionisation source (ESI), and
motility. Serum levels of MTL and GAS were measured using enzyme positive and negative ions were alternately scanned. The spray voltage
linked immunosorbent assay (ELISA) according to the manufacturer’s in the negative mode and positive mode were 3800 V and 3200 V
instructions (USCN Life Science Inc., Wuhan, China). respectively. The capillary temperature is 350 ◦ C. The scan mode was
full scan/data-dependent two-stage scan (full scan/ddMS2) under a scan
range of 100–1500 Da. Furthermore, the sheath gas and the auxiliary gas
2.6. HE staining were 35 arb and 15 arb respectively. Low, medium and high collision
energy were used in MS2 stage. The collision energy in the positive ion
The stomachs and duodenum were removed and immersed in 4 % mode was 30 V, 40 V, 50 V. The collision energy in the negative ion
formalin for 72 h at 4 ◦ C. Subsequently, stomach and duodenum tissues mode was 30 V, 50 V, 70 V. The resolution of primary mass spectrometry
were embedded in paraffin, and sectioned with a cryostat for HE staining was full scan 70,000 FWHM (full width at half maximum), and the
and histological evaluation. resolution of secondary mass spectrometry was MS/MS 17,500 FWHM.
At the beginning of the analysis, the precision and reproducibility
2.7. Metabolomic analysis were both evaluated by injecting the QC samples six times. The QC
sample was also injected every six samples during acquisition to mea­
2.7.1. Sample preparation sure stability.
For this, 50 μL serum samples were thawed and centrifuged at
12,000 rpm (5 min at 4 ◦ C) to remove particles. The pooled quality 2.7.3. Data preprocessing and analysis
control (QC) samples were made by mixing 10 μL aliquots from each The acquired raw data from Xcalibur were imported into Analysis
serum sample. Take serum samples or QC samples, add three times Base File Converter software for format conversion. The conversed data
acetonitrile, vortex for 3 min, ultrasonic extract for 10 min, vortex for were transferred into MSDIAL software for selection, deconvolution and
1 min, centrifuge at 800 rpm (10 min at 4 ◦ C) to obtain the supernatant.

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R. Liu et al. Journal of Chromatography B 1226 (2023) 123676

alignment, which includes retention time, m/z values, peak intensity concerned with FD.
and m/z. Peak filtering was carried out using the ‘80 % rule’ to reduce The common targets were obtained by intersecting FD-related targets
missing value input. Finally, the data containing compound ID number, and compounds-related targets based on the Jvenn website (https://
group type and normalised value was imported into SIMCA14.1 soft­ jvenn.toulouse.inra.fr/app/example.html), which were potential tar­
ware (UmetricsAB, Umeå, Sweden) [44] for further analysis. gets for XPHC in treating FD.
PCA DModX plot was constructed to exclude outliers. Next, principal
component analysis (PCA) was adopted to visualise the clustering of the 2.8.4. Gene ontology and KEGG pathway enrichment analysis
samples, which is an unsupervised multivariate pattern recognition The Database for Annotation, Visualisation and Integrated Discovery
analysis. Then, orthogonal partial least squares discriminant analysis (DAVID, https://david-d.ncifcrf.gov/content.jsp?file=update.html) was
(OPLS-DA) was employed to discriminate serum samples between the used for gene ontology (GO) and Kyoto Encyclopedia of Genes and Ge­
CC group and the intervention groups (FD; FD + H; FD + L; FD + Dom). nomes (KEGG) enrichment analysis. GO analysis includes biological
The 200 permutation test was used to evaluate the accuracy of the OPLS- process (BP), molecular function (MF) and cell component (CC). The
DA model. OPLS-DA was used for screening the metabolites with vari­ analysis was performed via Origin 2021 and Office 2019.
able importance in projection (VIP) value > 3, and Student’s t-test was
performed to select differential ions between each groups. In all cases, P 2.8.5. Construction of network diagram
value < 0.05 was considered to be statistically significant [45]. Then The protein–protein interaction (PPI) network was accomplished
differential metabolites were identified by compared the spectrum ob­ based on the STRING database (https://www.string-db.org/) and
tained from Xcalibur and the standard spectrum under different collision Cytoscape 3.7.1 platform [46]. Betweenness centrality (BC), closeness
energies. The standard spectrumes were provided by mzCloud databases centrality (CC) and degree centrality (DC) were selected to assess the
(https://www.mzcloud.org/) and the Human Metabolome database topological features of the PPI network. The threshold values of the hub
(HMDB, https://hmdb.ca/spectra/ms/search), whose molecular weight genes in the network analysis were the corresponding median values of
tolerance is qualified (ppm < 5). Differential metabolites were identified each parameter [47]. The ‘compound-hub gene-pathway’ network was
by compared the predicted spectrum with the spectrum obtained from also constructed using the Cytoscape 3.7.1 platform.
Xcalibur under different collision energies. MetaboAnalyst 5.0 platform
(https://www.metaboanalyst.ca/) was adopted to analysis the relative 2.9. Gene-metabolite interaction network
metabolic pathways which were associated with therapeutic mecha­
nisms of XPHC improves FD. The ‘gene-metabolite’ interaction network build a bridge between
the result of network pharmacology and metabolomics, also visualise
2.8. Network pharmacology construction the interactions between functional metabolites and key genes based on
the MetaboAnalyst 5.0. The associations of bioactive compounds and
2.8.1. Identification of XPHC components in the decoction liquid and blood human genes were extracted from the STRING website, which were
The 5 µL pre-processed decoction liquid of XPHC, CA, LRT, SBL and highly confident interactions. Then, the key genes screened in network
PN were analysed based on the UPLC-QE-Orbitrap-MS system. pharmacology and different metabolites from metabolomics were im­
Furthermore, the six rats were given continuous administration for ported into MetaboAnalyst 5.0 for network interaction analysis.
1 week, and then fasted for 12 h at the end of the trial, whilst having free
access to water. After the last dose, the drug-contained plasma was 2.10. Molecular docking
sampled from the tail of rats at 15 min, 30 min, 1 h, 2 h, 4 h and 6 h.
Pooled samples were made by mixing 50 μL aliquots from each sample. The Molecular docking was performed on Maestro 11.8 software.
Pre-preparation of drug-contained sample was identical to that of the The 3D structure file of the active compounds was download from the
metabolomics analysis. A 5 µL sample was then taken for the UPLC-QE- Pubchem Database (https://pubchem.ncbi.nlm.nih.gov/), which were
Orbitrap-MS analysis. And instrument method was similar to that of the used as small molecule ligands for molecular docking. The LigPrep panel
metabolomics analysis except for the gradient elution (0–1 min: 5 % B; was used to implement a variety of ligand preparation jobs, which
1–20 min: 5–98 % B; 20–21 min: 98–98 % B; 21–23 min: 98–5 % B; mainly defines ionisation states and stereoisomers. The protein crystal
23–26 min: 5 % B). structure was download from Maestro based on PDB ID, and optimised
under the construction of the Protein Preparation Wizard. Furthermore,
2.8.2. Collection predicted targets of bioactive compounds the binding site of the original ligand in the protein was defined as the
The compounds found in the plasma are theoretically considered as mating pocket, which will be used as the centre and size of the docking
potentially bioactive ingredients. The 2D structure of bioactive com­ pocket. Finally, the original ligand and the prepared small molecule
pounds were downloaded from PubChem database (https://pubchem. structure were simultaneously docked with the protein under the stan­
ncbi.nlm.nih.gov/) and submitted to Swiss Target Prediction (http dard precision (SP) mode.
s://swisstargetprediction.ch/) for potential targets. The targets with
probability > 0 were included. 3. Results
“The compounds found in the plasma are theoretically considered as
3.1. Improvement in functional dyspepsia with Xiaopi Hewei Capsule
bioactive ingredients” into “The compounds found in the plasma are
theoretically considered as potentially bioactive ingredients”
As shown in Fig. 3, before administration, gastric emptying body,
small intestine propulsion rate, the serum levels MTL and GAS of mice in
2.8.3. Targets of functional dyspepsia the model group was lower than that of mice in the control group
The targets of FD were collected by integrating the multi-source (P < 0.01). After treatment for one weeks, gastric emptying body and
databases. Databases used in this study were as follows: the Gene­ small intestine propulsion, the serum levels MTL and GAS of mice of
Cards database (https://www.genecards.org/), the Therapeutic Target mice in the FD + XHPC-H, FD + XHPC-L and FD + Dom groups were
Database (TTD, https://db.idrblab.org/ttd/) and Online Mendelian In­ increased, and the difference was statistically significant. Additionally,
heritance in Man database (OMIM, https://omim.org/). ‘Functional the improvement effect of the high dose was better than that of low dose
dyspepsia’ was the keyword during retrieving the three databases and group, and close to that of the domperidone group to some extent.
Homo sapiens targets were screened for further study. Additionally, FD- In order to further explore the pathological state of FD mice, the
related targets were aggregated based on experimental literature stomach and duodenum of different groups were stained and observed.

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R. Liu et al. Journal of Chromatography B 1226 (2023) 123676

Fig. 3. XPHC has effect on the symptoms of FD mice. Changes of the gastric emptying rate (A), the small intestinal propulsion rate (B), serum gastrin (C) and serum
motilin (D). All the data were expressed as mean ± SD. #p < 0.01, compared with control group; *p < 0.05, **p < 0.01, compared with FD group via ANOVA based
on SPSS 22.0.

The cell morphology and structure in each model group were complete, that the method repeatability, instrument precision and samples sta­
the cells were neatly arranged, and there was no inflammatory infil­ bility satisfied the analytical requirements.
tration or glandular epithelial disease, which is consistent with a clinical The representative BPI diagrams of different groups in negative ion
diagnosis based on the criteria of Rome III without organic disease. The mode and positive ion mode are shown in Fig. S2. PCA DModX diagram
details are shown in Fig. 4. These changes indicate that XPHC has the constructed in SIMCA 14.1 software can be used to diagnose serum
potential to improve the symptoms of FD in mice. samples five groups and evaluate the reliability of datas, As shown in
Fig. 5A and Fig. 5B. The original data of the five groups are within the
3.2. Metabolomic profile confidence interval and can be included for further statistical analysis.
Fig. 5C and 5D demonstrate that samples from different groups exist
The PCA score plots and the representative basic peak ion (BPI) independently, and samples within the same group can be gathered
chromatograms of QC samples were showed in the Fig. S1, indicating together well.

Fig. 4. Morphological observation of stomach and duodenum.

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R. Liu et al. Journal of Chromatography B 1226 (2023) 123676

Fig. 5. PCA DModx plots of total serum samples for analysis validation under the negative ion mode and positive ion mode (A and B). 3D Score plots from PCA
models for prediction under the negative ion mode and positive ion mode (C and D).

Furthermore, the OPLS-DA supervised pattern recognition method 3.4. Network pharmacology
was applied to identify the overall metabolic differences between two
groups, which was confirmed by the 200 permutation test. As shown in 3.4.1. Identification of bioactive compounds
Fig. 6A, significantly separated clusters appeared between the CC group The network pharmacology was conducted to further explore the
and the FD group. Fig. 6B shows that the values of R2 and Q2 on the right mechanisms of XPHC against FD. The BPI diagrams of the decoction
are higher than the values on the left. Additionally, the intersection liquid of XPHC, CA, LRT, SBL, PN, and drug-containing plasma were
value of the regression line of Q2 and the vertical axis was negative. acquired based on UPLC-QE-Orbitrap-MS, and shown in Fig. 9. In total,
Thus, it is possible to predict potential metabolites based on the OPLS- 96 compounds in decoction liquid of XPHC were predicted or identified
DA model. S-plots diagrams gave information of VIP value (Fig. 6C), through literature comparison and standard compound data compari­
which was used to identify characteristic metabolites in five groups. son, and the results were shown in Table S2. The source of 96 com­
Moreover, the score plots of OPLS-DA models and 200 permutation tests pounds were determined by investigating the MS spectrum of CA, LRT,
for prediction of the FD, FD + XPHC-H, FD + XPHC-L and FD-Dom SBL and PN respectively. A further analysis was conducted based on the
groups are shown in Fig. S3. MS spectrum of drug-containing plasma to explore effective component
of XPHC in vivo. Finally, there are 27 compounds screened from plasma
3.3. Differential metabolite analysis under positive and negative ion mode; the results were shown in
Table S3.
By setting the screening threshold as VIP ≥ 3 and p < 0.05, we ob­
tained 20 differential metabolites based on MS/MS spectrum informa­ 3.4.2. Construction of the network diagram
tion and the HMDB database (Table 1). The changing trends of 20 Based on the 27 compounds absorbed into plasma, 765 compound-
different metabolites in different groups are visualised using the heat related targets were obtained through the Swiss Target Prediction
map and scatter plots (Fig. S4 and Fig. 7). XPHC can upregulate 13 website. In total, 3,033 FD-related targets were collected through the
biomarkers, and downregulate 7 biomarkers, indicating that the meta­ GeneCards, TTD and OMIM databases; 352 common targets were ob­
bolic perturbations of FD can be partly improved by XPHC. Metabolic tained by intersecting FD-related targets and compound-related targets
pathway analysis was performed by importing these differential me­ based on the Jvenn website, as shown in Fig. 10A. Furthermore, GO and
tabolites into MetaboAnalyst 5.0 platform to search for the potential KEGG pathway enrichment analysis were accomplished based on the
mechanisms. A total of 13 metabolic pathways were screened out, such 352 potential targets via the DAVID system. Thus, 1,471 GO terms (BP:
as sphingolipid metabolism, citrate cycle (TCA cycle), glyoxylate and 1077; CC: 137; MF: 257) and 191 KEGG pathways were obtained, and
dicarboxylate metabolism, arginine and proline metabolism and so on the top 20 items of GO and KEGG analysis were visualised through origin
(Fig. 8 and Table S1). 2021 software (Fig. 10B and 10C, respectively). Nine hub genes (ALB,
GAPDH, AKT1, TP53, VEGFA, MAPK3, TNF, EGFR, SRC) were screened

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R. Liu et al. Journal of Chromatography B 1226 (2023) 123676

Fig. 6. Score plots from OPLS-DA models (A), 200 permutation tests (B) and S-plots diagrams (C) for prediction of CC group and FD group under the negative ion
mode and positive ion mode respectively.

out by taking values of BC, CC and DC into consideration; the topological into MetaboAnalyst 5.0 for network interaction analysis, the result was
PPI network was displayed in Fig. 10 D. Detailed information on the 9 shown in Fig. 11A. The gene-metabolite interaction network consists of
hub genes was shown in Table 2. twenty-seven targets and nine metabolites, where squares represent
The relationship between 27 active components (blue nodes), 352 differential metabolites and circles represent target genes. Based on the
hub genes (cyan nodes) and the top 20 KEGG pathways (purple nodes) value of degree and betweenness, eicosapentaenoic acid, citric acid and
are presented in the ‘compound-gene-pathway’ network map prepared L-leucine can be considered as critical metabolites; detailed information
using Cytoscape (Fig. S5). The top ten compounds were ferulic acid, was shown in Table S4. Eicosapentaenoic acid has a close interaction
pyrogallic acid, kaempferol, caudatin, ginsenoside Rg1, ginsenoside Re, with the eighteen targets in the network, citric acid has a close associ­
2,4-dihydroxyacetophenone, metaplexigenin, ginsenoside Rd, and gin­ ation with 14 targets, and L-leucine has a close interaction with four
senoside Rh1 in descending order of degree, demonstrating the crucial targets. It was surprising to find that four hub genes (ALB, EGFR, TNF
roles of these components in treating FD. Representative ESI-MS spectra and SRC) were involved in the construction of the network map.
and ESI-MSn spectra of 10 compounds were shown in Fig. S6. Moreover, the metabolite-metabolite interaction network contributes to
highlighting the potential functional relationships between a wide range
of annotated metabolites. Therefore, the metabolite-metabolite inter­
3.5. Gene-metabolite interaction network action network analysis had been conducted based on twenty different
metabolites; the results were shown in Fig. 11B. Citric acid, L-leucine
The 352 common targets and 20 different metabolites were imported

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R. Liu et al. Journal of Chromatography B 1226 (2023) 123676

Table 1
The information of different metabolites in serum profiles.
Ion No. tR/ Molecular Measured value (m/ Theoretical value (m/ Metabolites HMDB ID VIP Up/
mode min Formula z) z) Down

ESI+ 1 1.20 C5H11NO2 118.0865 118.0863 5-Aminopentanoic acid HMDB0003355 11.66 ↓

2 1.26 C4H9N3O2 132.0767 132.0768 Creatine HMDB0000064 13.74 ↓
3 1.40 C6H13NO2 132.1022 132.1019 Leucine HMDB0000687 12.34 ↓
4 1.40 C10H9NO 160.0756 160.0757 Indoleacetaldehyde HMDB0001190 9.83 ↑
5 1.87 C5H4N4O2 153.0406 153.0407 Xanthine HMDB0000292 3.72 ↓
6 9.89 C18H39NO2 302.3050 302.3054 Sphinganine HMDB0000269 1.50 ↑
7 12.72 C25H52NO7P 510.3552 510.3554 LysoPE(20:0/0:0) HMDB0011511 3.89 ↓
8 14.91 C27H56NO7P 538.3862 538.3867 LysoPE(22:0/0:0) HMDB0011520 8.24 ↓
ESI- 9 1.25 C6H8O7 191.0190 191.0186 Citric acid HMDB0000094 3.65 ↓
10 1.40 C4H6O5 133.0132 133.0131 Malic acid HMDB0000744 5.14 ↓
11 1.40 C5H4N4O4 183.0152 183.0149 5-Hydroxyisourate HMDB0030097 5.56 ↑
12 5.55 C7H13NO3 158.0814 158.0812 2-Methylbutyrylglycine HMDB0000339 25.54 ↑
13 5.74 C9H9NO3 178.0504 178.0499 Hippuric acid HMDB0000714 15.18 ↑
14 6.15 C10H11NO3 192.0660 192.0655 Phenylacetylglycine HMDB0000821 8.61 ↑
15 6.28 C7H8O4S 187.0064 187.0060 p-Cresol sulfate HMDB0011635 29.48 ↑
16 11.63 C18H32O3 295.2278 295.2268 13-Hydroxyoctadecadienoic HMDB0004667 6.53 ↓
acid
17 12.14 C18H34O3 297.2435 297.2424 10-Oxooctadecanoic acid HMDB0030980 3.77 ↓
18 13.99 C23H48NO7P 480.3095 480.3085 LysoPE(18:0/0:0) HMDB0011130 2.49 ↓
19 14.13 C20H30O2 301.2175 301.2162 Eicosapentaenoic acid HMDB0001999 6.09 ↓
20 14.70 C22H32O2 327.2330 327.2319 Retinol acetate HMDB0035185 14.43 ↓

Fig. 7. Regulatory characteristics of 20 differential metabolites in different groups. All the data were expressed as mean ± SD. #p < 0.05, ##p < 0.01, compared
with control group; *p < 0.05, **p < 0.01, compared with FD group via ANOVA based on SPSS 22.0.

and eicosapentaenoic acid were ranked highly according to the value of the metabolites citric acid, L-leucine and eicosapentaenoic acid were
degree and betweenness, which occupy an extremely important position considered as crucial biomarkers and have crucial biological signifi­
in the metabolite interaction network. Detailed information was shown cance in the treatment of FD with XPHC.
in Table S5. The comprehensive analysis of gene-metabolite interaction
network and metabolite-metabolite interaction network indicate that

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R. Liu et al. Journal of Chromatography B 1226 (2023) 123676

Fig. 8. Bubbles map of metabolism pathway of the potential differential metabolites of XPHC in intervention of FD mice (A). The network diagram of metabolism
pathway and differential metabolites (B).

3.6. Molecular docking inflammation and Hp infection are currently considered as the key
pathological mechanisms of FD. Animal models have been widely used
The binding interactions between the top ten prototype components to study the pathological mechanism of FD, but widely recognized
in the blood and 9 hub targets were predicted by molecular docking models and drugs for FD have not yet been developed. Iodoacetamide
based on Maestro 11.8 software, which could lay the foundation to treatment is a classical and comprehensive method used internationally
explore the mechanism of XPHC in treating FD. As displayed in Table 3, to develop the FD animal model [50,51]. Iodoacetamide has been re­
the binding energies were computed to evaluate the binding affinities of ported to cause mild gastritis, along with impairments of gastric sensory
the 10 compounds with the 9 proteins. It is known that when a ligand and motor function [52]. The establishment of the FD model using tail-
has a lower binding free energy, this indicates a higher binding affinity clamping stimulation is more popular in most domestic researchers, and
to its receptor. The binding energies of the 10 compounds with the 9 is being accepted by foreign scholars [53]. Tail-clamping irritates ani­
receptors ranged from − 2.092 to − 11.286 kcal/mol, suggesting that mals and causes fighting, which may lead to anxiety-like behaviors and
these compounds may have potential therapeutic effects in FD. Among delayed gastric emptying [53,54].Furthermore, alternate-day fasting,
them, SRC and ALB have the lowest binding energy with 2,4-dihydrox­ water-immersion restraint stress and swimming-induced fatigue are also
yacetophenone. The combination of EGFR is the best with kaempferol. commonly used in the development of FD models [55,56]. Among them,
Additionally, TNF have the best combination with ginsenoside Rg1. The alternate-day fasting simulates the characteristics of FD patients with
2D action mode graph of the representative compounds and related irregular diet. Water immersion restraint stress can cause intestinal
receptors were shown in Fig. S7. stress and inflammatory reaction in laboratory animals. Swimming-
induced fatigue is reported to increase plasma adrenocorticotrophic
4. Discussion hormone (ACTH) and cortisol levels. It also induces delay in gastric
emptying [56]. Different modelling methods have different advantages.
So far, people in >140 countries or regions worldwide have used Having the characteristics of a high self-healing rate, short duration and
traditional medicine (including traditional Chinese medicine or ethnic single characteristics, single-factor animal models are not able to fully
medicine) in disease prevention and treatment [48]. However, tradi­ simulate the complex aetiology, pathogenesis and symptoms of FD.
tional drugs are generally a mixture of multiple compounds [49]. There Therefore, some scholars use double-factor and multi-factor stimulation
is an absence of guiding academic ideas and method innovations to methods to establish FD animal models, which avoid high self-healing
clarify the active ingredients, biological activities and pharmacological based on a single stimulus, and reflect the real situation of disease
mechanisms, which restricts in-depth research and further development characteristics. In this work, iodoacetamide treatment, tail-clamping
of ethnic traditional medicine [35]. Xiaopi Hewei Capsule (XPHC), a and alternate-day fasting were used in combination to build a FD
classical folk ‘Miao minority’ medicine, has the effect of improving model with the characteristics of visceral hypersensitivity, anxious and
stomach heat, pain, heartburn and noisiness [25]. Although there are irregular diet [52].
corresponding clinical reports of XPHC in treating FD, it has not been Decreases in the gastric emptying rate and small intestine propulsion
widely used due to the unexplained treatment mechanism [11]. rate caused by abnormal gastrointestinal motility are typical charac­
teristics in patients with FD. The gastric emptying rate and the small
4.1. FD animal model analysis intestine propulsion rate were significantly reduced in the FD group
compared with the control group, which were improved after the
The etiology of patient with FD is complex and its pathogenesis has intervention with different doses of XPHC and domperidone. Addition­
not been fully defined. Disrupted gastrointestinal motility, visceral hy­ ally, MTL is a brain-gut peptide that is closely related to gastric motility
persensitivity, psychological disease, irregular diet, intestinal and gastric emptying. GAS distribution in the gastric antrum can

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R. Liu et al. Journal of Chromatography B 1226 (2023) 123676

Fig. 9. The BPI diagrams of CA, LRT, SBL, PN, the decoction liquid of XPHC and drug-containing plasma under the negative ion mode and positive ion mode.

stimulate gastric acid secretion and enhance gastric antral motility. The metabolites. Combined with gene-metabolite interaction network and
serum levels of GAS and MTL were significantly decreased in the FD metabolite-metabolite interaction network, four hub genes (ALB, EGFR,
group compared with the control group. XPHC and domperidone TNF and SRC) and three key serum biomarkers (citric acid, L-leucine and
treatment markedly increased the levels of GAS and MTL compared with eicosapentaenoic acid) were considered to have important biological
the FD group. In summary, a FD model was successfully established significance.
under the stimulation of three factors, and the therapeutic effect of Serum ALB has a number of physiological functions such as
XPHC on mice with FD was also observed. increasing circulating blood volume, anti-inflammatory and anti-
oxidant [57]. A reduction in serum ALB is closely related to inflamma­
tory bowel disease [58]. The serum level of ALB usually reflects the
4.2. Therapeutic mechanism analysis
nutritional status of the organism [59]. Clinical studies have pointed out
that patients with FD show a significant decrease in serum ALB content
The gene-metabolite interaction network consists of 27 targets and 9
due to abnormal digestion, malabsorption and insufficient energy intake
metabolites, which shows the interactions between genes and

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R. Liu et al. Journal of Chromatography B 1226 (2023) 123676

Fig. 10. The venn diagram of the potential targets of XPHC in treating FD(A); Top 20 items of GO enrichment analysis (B); Top 20 pathways of KEGG enrichment
analysis (C); The topological PPI network (D).

Table 2
Hub gene information of XPHC treating FD in network pharmacology.
No. Gene symbol Uniprot ID Protein name Key parameters in topological analysis

Betweenness centrality Closeness centrality Degree

1 ALB P02768 Albumin 0.0688 0.6992 201

2 GAPDH P04406 Glyceraldehyde-3-phosphate dehydrogenase 0.0533 0.6992 201
3 AKT1 Q96B36 Proline-rich AKT1 substrate 1 0.0434 0.6923 199
4 TP53 P04637 Cellular tumor antigen p53 0.0512 0.6842 193
5 VEGFA P15692 Vascular endothelial growth factor A 0.0338 0.6635 174
6 MAPK3 P27361 Mitogen-activated protein kinase 3 0.0285 0.6549 169
7 TNF P01375 Tumor necrosis factor 0.0299 0.6500 164
8 EGFR P00533 Epidermal growth factor receptor 0.0281 0.6500 164
9 SRC P12931 roto-oncogene tyrosine-protein kinase Src 0.0251 0.6393 154

Fig. 11. Interaction network between target genes and differential metabolites identified in serum samples (A); differential metabolite-metabolite interaction
network identified in serum samples (B).

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R. Liu et al. Journal of Chromatography B 1226 (2023) 123676

Table 3
Molecular docking results of 10 prototype components absorbed into the bloodr from XPHC.
Compounds ALB GAPDH AKT1 TP53 VEGFA MAPK3 TNF EGFR SRC
2BXF 6YND 4EJN 6GGA 4KZN 6GES 5MU8 5D41 3D7T

Ligand − 3.984 − 3.054 − 4.888 − 4.569 − 3.465 − 3.942 − 5.048 − 3.456 5.607
Ferulic acid − 5.226 − 3.286 − 5.3 − 5.274 − 4.738 − 4 − 4.964 − 9.186 5.252
Kaempferol − 5.88 − 4.783 − 8.566 − 7.628 − 5.791 − 6.669 − 6.464 − 9.727 7.245
Pyrogallic acid − 5.436 − 5.081 − 6.113 − 6.177 − 5.925 − 5.702 − 5.618 − 6.596 6.007
Caudatin − 3.535 − 2.251 – − 4.713 − 2.108 − 2.092 − 6.655 − 6.916 6.166
Ginsenoside Rg1 – – − 11.286 − 4.627 − 3.485 − 3.152 − 6.954 − 3.032 6.738
Ginsenoside Re – – – − 3.387 − 4.597 − 3.351 − 5.744 − 5.491 5.974
2,4-dihydroxyacetophenone − 6.151 − 4.303 − 6.052 − 6.793 − 5.973 − 5.278 − 6.049 − 9.706 7.293
Ginsenoside Rd – – – – − 3.763 − 4.77 − 5.682 − 5.665 5.372
Metaplexigenin − 3.033 – − 4.077 − 2.984 − 4.875 − 3.922 − 5.588 − 4.232 6.954
Ginsenoside Rh1 − 3.006 − 3.006 − 9.779 − 3.978 − 3.144 − 3.431 − 5.637 − 4.952 5.602

[60]. Research has suggested that SRC may promote the occurrence of eicosapentaenoic acid, citric acid and hippuric acid. SRC mediates the
intestinal inflammation by mediating the release of inflammatory fac­ process of the tricarboxylic acid cycle by regulating the metabolism of
tors [61]. A further investigation showed that SRC participates in citric acid.EGFR regulates the metabolism of eicosapentaenoic acid and
cytokine regulation, cell migration and proliferation by mediating the 13-hydroxyoctadadienoic acid. TNF is involved in the sphingolipid
ErbB signalling pathway [62]. In addition, ALB and SRC may promote metabolism pathway. According to the “compound-key gene-KEGG
the occurrence of intestinal inflammation by mediating the release of pathway” network interaction, among the top 10 compounds caudatin
inflammatory factors. EGF (epidermal growth factor) and EGFR had and 2,4-dihydroxyacetophenone are closely related to ALB. Represen­
been proved to play a crucial role in the proliferation and repair of tative compounds acting on SRC are pyrogallic acid, ginsenoside Re. Key
gastric mucosal cells [63,64]. The TNF gene is emphasised in research on compounds that communicate with EGFR are pyrogallic acid, ferulic
duodenal barrier damage and intestinal inflammation in FD mice acid, metaplexigenin, kaempferol, 2,4-dihydroxyacetophenone and
[65,66]. TNF participates in inflammation by mediating the NF-κB sig­ ginsenoside Rg1. The compound that acts with TNF is metaplexigenin.
nalling pathway and the Toll-like receptor signalling pathway, which Caudatin and 2,4-dihydroxyacetophenone regulate the ALB gene, and
are activated in animal models of FD [67]. Thus, the selected key genes mediate energy metabolism and lipid metabolism by affecting the levels
are involved in the repair process of FD by resisting inflammation and of citric acid, and eicosapentaenoic acid. Pyrogallic acid and meta­
promoting the repair of gastric mucosa cells. plexigenin regulate the EGFR gene, and mediate lipid metabolism and
Citric acid participates in the tricarboxylic acid cycle (TCA) meta­ intestinal inflammation. Based on the molecular docking results in
bolic pathway and Glyoxylate and dicarboxylate metabolism pathway, Table 3, it can be seen that compared with the original ligand, the four
which are important pathways of energy metabolism. The TCA cycle is hub genes of ALB, EGFR, TNF and SRC have better binding ability to 10
the hub of the metabolism of carbohydrates, lipids and amino acids. active compounds. It is speculated that the active compounds can play a
Citric acid is the first compound of the TCA cycle, which releases energy role in treating FD by acting on the core targets.
through a series of dehydrogenation and decarboxylation reactions. In conclusion, The regulation of genes and metabolites in the
Glyoxylate and dicarboxylate metabolism pathway The serum content of development of FD is seen to be consistent. The 10 key active com­
citric acid in FD mice was significantly lower than in control mice, and pounds of XPHC act on 4 core targets, interfere with endogenous me­
the TCA cycle was abnormal, leading to insufficient energy supply. tabolites, and affect energy metabolism, amino acid metabolism, lipid
Abnormal metabolism of amino acids and lipids will indirectly affect the metabolism, inflammatory reactions and mucosal repair. The core gene
TCA cycle. Additionally, L-leucine is involved in valine/leucine/ builds a bridge between differential metabolites and active compounds.
isoleucine biosynthesis, valine/leucine/isoleucine degradation and The active compounds of XPHC affect gene expression and return the
aminoacyl-tRNA biosynthesis metabolic pathway, whih are related with changed metabolites to normal levels through specific metabolic path­
amino acid metabolism. Scientists have pointed out that a decreased ways. This process is beneficial for maintaining homeostasis and pro­
level of serum L-leucine is one of the characteristics of serum meta­ moting recovery in patient with FD. Mutual regulation between active
bolism in FD patients compared with healthy controls, which is related compounds-genes-metabolites is a potential way for Xiaopi Hewei
to metabolic consumption caused by reduced appetite and malnutrition Capsules to treat functional dyspepsia.
[68,69]. Similarly, this experiment confirmed that the serum level of L-
leucine in FD mice was significantly lower than that of control mice. 5. Conclusion
Eicosapentaenoic acid participates in biosynthesis of unsaturated fatty
acids metabolic pathway. Eicosapentaenoic acid is an essential unsatu­ In summary, serum metabolomics combined with network pharma­
rated fatty acid converted from ingested food. Studies have confirmed cology showed that XPHC provided protection against FD via a target
that eicosapentaenoic acid has anti-tumour, anti-inflammatory and cell network and multiple pathways. Ten active compounds (ferulic acid,
membrane stabilisation effects [70,71]. Compared with the control pyrogallic acid, kaempferol, caudatin, ginsenoside Rg1, ginsenoside Re,
group, the serum content of eicosapentaenoic acid of FD mice was 2,4-dihydroxyacetophenone, metaplexigenin, ginsenoside Rd, and gin­
significantly lower, indicating that the eicosapentaenoic acid synthesis senoside Rh1), four hub genes (ALB, EGFR, TNF and SRC) and three key
process was blocked. In addition, sphingolipid metabolism is one of the biomarkers (citric acid, L-leucine and eicosapentaenoic acid) respon­
crucial metabolic pathway obtained according to Fig. 8. Sphinganine is sible for maintaining homeostasis and promoting recovery in patient
one of the metabolites of sphingolipid, with anti-inflammatory and skin with FD. The integrated network also revealed that pathway regulation
moisturising effects [57]. Sphinganine metabolism and biosynthesis of by XPHC,mainly including energy metabolism, amino acid metabolism,
unsaturated fatty acids metabolic pathway are key parts of lipid meta­ lipid metabolism, inflammatory reactions and mucosal repair. Mean­
bolism. It can be concluded that the serum biomarkers are involved in while, our results strongly support the current understanding of the role
the development process of FD by regulating energy metabolism, amino of XPHC in treating FD, which may provide new ideas and reference for
acid metabolism and lipid metabolism. further scientific research. However, the organism is complex, and the
Typically, as the centre of the gene-metabolite interaction network interaction between metabolites and genes has not yet been definitively
diagram, ALB participates in the metabolic regulation of concluded. Thus, using network pharmacology or metabolomics can

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R. Liu et al. Journal of Chromatography B 1226 (2023) 123676

only predict the potential mechanism of XPHC in FD. The results ob­ [10] H. Sunxiang, X. Zemin, W. Bo, Xiaopi Hewei Capsules Treatment on 32 Cases with
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